Anti-siglec-8 antibodies and methods of use thereof

ABSTRACT

The invention provides humanized anti-Siglec-8 antibodies and their use in treating and preventing eosinophil-mediated disorders and/or mast cell-mediated disorders, as well as compositions and kits comprising the humanized anti-Siglec-8 antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.16/416,085, filed May 17, 2019, which is a continuation application ofU.S. patent application Ser. No. 15/372,268, filed Dec. 7, 2016, nowabandoned, which is a continuation application of U.S. patentapplication Ser. No. 14/565,370, filed Dec. 9, 2014, now U.S. Pat. No.9,546,215, issued Jan. 17, 2017, which claims priority to U.S.Provisional Patent Application No. 61/913,891, filed Dec. 9, 2013, thecontents of each of which are incorporated herein by reference in theirentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 701712000103SeqList.txt,date recorded: Jan. 26, 2022, size: 117,399 bytes).

FIELD OF THE INVENTION

This invention relates to anti-human Siglec-8 antibodies and methods oftreating or preventing a disease mediated by cells expressing Siglec-8.

BACKGROUND OF THE INVENTION

Siglecs (sialic acid-binding immunoglobulin-like lectins) aresingle-pass transmembrane cell surface proteins found predominantly onleukocytes and that are characterized by their specificity for sialicacids attached to cell-surface glycoconjugates. The Siglec familycontains at least 15 members that are found in mammals (Pillai et al.,Annu Rev Immunol., 2012, 30:357-392). These members includesialoadhesion (Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3), myelinassociated glycoprotein (Siglec-4), Siglec-5, OBBP1 (Siglec-6), AIRM1(Siglec-7), SAF-2 (Siglec-8), and CD329 (Siglec-9). Siglec-8, a memberthat is expressed in humans but not in mouse, was first discovered aspart of efforts to identify novel human eosinophil proteins. In additionto expression by eosinophils, it is also expressed by mast cells andbasophils. Siglec-8 recognizes a sulfated glycan, i.e., 6′-sulfo-sialylLewis X or 6′-sulfo-sialyl-N-acetyl-S-lactosamine, and contains anintracellular immunoreceptor tyrosine-based inhibitory motif (ITIM)domain shown to inhibit mast cell function.

Along with mast cells, eosinophils can promote an inflammatory responsethat plays a beneficial functional role such as controlling an infectionat a specific tissue site. During an inflammatory response, apoptosis ofeosinophils can be inhibited through the activity of survival-promotingcytokines such as IL-3 and GM-CSF. However, an increase of activatedeosinophils that are not rapidly removed by apoptosis can result in therelease of eosinophil granule proteins at already inflamed sites whichcan damage tissue and cause inflammation to be further exacerbated.Several diseases have been shown to be linked to eosinophil activationsuch as Churg Strauss syndrome, rheumatoid arthritis, and allergicasthma (Wechsler et al., J Allergy Clin Immunol., 2012, 130(3):563-71).There is currently a need for therapies that can control the activity ofimmune cells involved in inflammation, such as the activity ofeosinophils and mast cells.

Previous studies have demonstrated that eosinophils undergo apoptosiswhen Siglec-8 is crosslinked with specific murine antibodies raisedagainst the extracellular portion of Siglec-8 (Nutku et al., Blood,2003, 336:918-24). These antibodies are described in U.S. Pat. Nos.8,207,305, 8,197,811, 7,871,612, and 7,557,191. However, there remains aneed for developing humanized anti-Siglec-8 antibodies that recognizehuman Siglec-8 with high affinity and specificity. Discovery of suchanti-Siglec-8 antibodies may allow for the development of treatment fordiseases mediated by the activity of eosinophils and/or mast cells.

All references cited herein, including patent applications, patentpublications, and scientific literature, are herein incorporated byreference in their entirety, as if each individual reference werespecifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

Provided herein are anti-Siglec-8 antibodies (including humanizedanti-Siglec-8 antibodies), compositions comprising thereof, and methodsof using the same.

In one aspect, provided herein is a humanized antibody that specificallybinds to a human Siglec-8, wherein the binding affinity and/or bindingavidity of the humanized antibody to a human Siglec-8 are higher thanthe binding affinity and/or binding avidity of antibody 2E2 and/orantibody 2C4 to the human Siglec-8. In some embodiments, the humanSiglec-8 is a dimer. In some embodiments, the human Siglec-8 comprisesan extracellular domain human Siglec-8 fused to a Fc region of animmunoglobulin. In some embodiments, the Fc region is a human IgG1 Fcregion. In some embodiments, the Fc region is a human IgG4 Fc region. Insome embodiments, the human Siglec-8 comprises the amino acid sequenceof SEQ ID NO:74.

In some embodiments, the humanized antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises (i) HVR-H1 comprising the amino acidsequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequence ofSEQ ID NO:63; and/or wherein the light chain variable region comprises(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii)HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and (iii)HVR-L3 comprising the amino acid sequence of SEQ ID NO:66. In someembodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:6; and/or a light chainvariable region comprising the amino acid sequence selected from SEQ IDNOs:16 or 21. In any of the embodiments herein, the antibody maycomprise a heavy chain Fc region comprising a human IgG Fc region. Infurther embodiments, the human IgG Fc region comprises a human IgG1 orIgG4. In some embodiments, the human IgG1 comprises the amino acidsequence of SEQ ID NO:78. In some embodiments, the human IgG4 comprisesthe amino acid sequence of SEQ ID NO:79. In any of the embodimentsherein, the antibody may comprise a heavy chain comprising the aminoacid sequence of SEQ ID NO:75; and/or a light chain comprising the aminoacid sequence selected from SEQ ID NOs:76 or 77. In some embodiments,the human IgG4 comprises the amino acid substitution S228P, wherein theamino acid residues are numbered according to the EU index as in Rabat.In any of the embodiments herein, the antibody may comprise a heavychain comprising the amino acid sequence of SEQ ID NO:87; and/or a lightchain comprising the amino acid sequence of SEQ ID NO:76. In someembodiments, the antibody has been engineered to improveantibody-dependent cell-mediated cytotoxicity (ADCC) activity. In someembodiments, the antibody comprises two heavy chains and wherein atleast one of the two or both heavy chains of the antibody isnon-fucosylated.

In another aspect, provided herein is a humanized antibody thatspecifically binds to a human Siglec-8, wherein the antibody has a Tm ofat least about 70° C. to at least about 72° C. in a thermal shift assay.In some embodiments, the antibody has a Tm at about 70° C., at about 71°C., or at about 72° C. in a thermal shift assay. In some embodiments,the antibody has the same or higher Tm as compared to a chimeric 2C4antibody. In some embodiments, the antibody has the same or higher Tm ascompared to an antibody having a heavy chain comprising the amino acidsequence of SEQ ID NO:84 and a light chain comprising the amino acidsequence of SEQ ID NO:85.

In some embodiments, the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises (i) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO:62, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:63; and/or wherein the light chain variable region comprises (i)HVR-L1 comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:66. In some embodiments,the antibody comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:6; and/or a light chain variable regioncomprising the amino acid sequence selected from SEQ ID NOs:16 or 21. Inany of the embodiments herein, the antibody may comprise a heavy chainFc region comprising a human IgG Fc region. In further embodiments, thehuman IgG Fc region comprises a human IgG1 or IgG4. In some embodiments,the human IgG1 comprises the amino acid sequence of SEQ ID NO:78. Insome embodiments, the human IgG4 comprises the amino acid sequence ofSEQ ID NO:79. In any of the embodiments herein, the antibody maycomprise a heavy chain comprising the amino acid sequence of SEQ IDNO:75; and/or a light chain comprising the amino acid sequence selectedfrom SEQ ID NOs:76 or 77. In some embodiments, the human IgG4 comprisesthe amino acid substitution S228P, wherein the amino add residues arenumbered according to the EU index as in Rabat. In any of theembodiments herein, the antibody may comprise a heavy chain comprisingthe amino acid sequence of SEQ ID NO:87; and/or a light chain comprisingthe amino acid sequence of SEQ ID NO:76. In some embodiments, theantibody has been engineered to improve antibody-dependent cell-mediatedcytotoxicity (ADCC) activity. In some embodiments, at least one or twoof the heavy chains of the antibody is non-fucosylated.

In yet another aspect, provided herein is a humanized antibody thatspecifically binds to a human Siglec-8, wherein the antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the aminoacid sequence selected from SEQ ID NOs:63 and 67-70; and/or wherein thelight chain variable region comprises (i) HVR-L1 comprising the aminoacid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acidsequence of SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acidsequence of SEQ ID NO:66 or 71. In some embodiments, the antibodycomprises a heavy chain variable region comprising the amino acidsequence selected from SEQ ID NOs:11-14; and/or a light chain variableregion comprising the amino acid sequence selected from SEQ IDNOs:23-24. In some embodiments, the heavy chain variable regioncomprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61,(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and(iii) HVR-H3 comprising the amino acid sequence selected from SEQ IDNO:63; and/or the light chain variable region comprises (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:66. In some embodiments,the antibody comprises a heavy chain variable region comprising theamino acid sequence selected from SEQ ID NO:6; and/or a light chainvariable region comprising the amino acid sequence selected from SEQ IDNO:16 or 21. In any of the embodiments herein, the antibody may comprisea heavy chain Fc region comprising a human IgG Fc region. In furtherembodiments, the human IgG Fc region comprises a human IgG1 or IgG4. Insome embodiments, the human IgG1 comprises the amino acid sequence ofSEQ ID NO:78. In some embodiments, the human IgG4 comprises the aminoacid sequence of SEQ ID NO:79. In some embodiments, the human IgG4comprises the amino acid substitution S228P, wherein the amino acidresidues are numbered according to the EU index as in Kabat. In someembodiments, the antibody has been engineered to improveantibody-dependent cell-mediated cytotoxicity (ADCC) activity. In someembodiments, at least one or two of the heavy chains of the antibody isnon-fucosylated.

In yet another aspect, provided herein is a humanized antibody thatspecifically binds to human Siglec-8, wherein the antibody comprises aheavy chain variable region comprising the amino acid sequence selectedfrom SEQ ID NOs:2-14; and/or a light chain variable region comprisingthe amino acid sequence selected from SEQ ID NOs:16-24. In someembodiments, the antibody has been engineered to improveantibody-dependent cell-mediated cytotoxicity (ADCC) activity. In someembodiments, at least one or two of the heavy chains of the antibody isnon-fucosylated.

In yet another aspect, provided herein is a humanized antibody thatspecifically binds to human Siglec-8, wherein the antibody comprises aheavy chain variable region comprising the amino acid sequence selectedfrom SEQ ID NOs:2-10; and/or a light chain variable region comprisingthe amino acid sequence selected from SEQ ID NOs:16-22. In someembodiments, the antibody has been engineered to improveantibody-dependent cell-mediated cytotoxicity (ADCC) activity. In someembodiments, at least one or two of the heavy chains of the antibody isnon-fucosylated.

In another aspect, provided herein is a humanized antibody thatspecifically binds to a human Siglec-8, wherein the antibody comprises aheavy chain variable region and a light chain variable region, wherein(a) the heavy chain variable region comprises: (1) an HC-FR1 comprisingthe amino acid sequence selected from SEQ ID NOs:26-29; (2) an HVR-H1comprising the amino acid sequence of SEQ ID NO:61; (3) an HC-FR2comprising the amino acid sequence selected from SEQ ID NOs:31-36; (4)an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62; (5) anHC-FR3 comprising the amino acid sequence selected from SEQ IDNOs:38-43, (6) an HVR-H3 comprising the amino acid sequence of SEQ IDNO:63, and (7) an HC-FR4 comprising the amino acid sequence selectedfrom SEQ ID NOs:45-46, and/or (b) the light chain variable regioncomprises: (I) an HC-FR1 comprising the amino acid sequence selectedfrom SEQ ID NOs:48-49; (2) an HVR-H1 comprising the amino acid sequenceof SEQ ID NO:64; (3) an HC-FR2 comprising the amino acid sequenceselected from SEQ ID NOs:51-53; (4) an HVR-H2 comprising the amino acidsequence of SEQ ID NO:65; (5) an HC-FR3 comprising the amino acidsequence selected from SEQ ID NOs:55-58; (6) an HVR-H3 comprising theamino acid sequence of SEQ ID NO:66; and (7) an HC-FR4 comprising theamino acid sequence of SEQ ID NO:60. In some embodiments, the antibodyhas been engineered to improve antibody-dependent cell-mediatedcytotoxicity (ADCC) activity. In some embodiments, at least one or twoof the heavy chains of the antibody is non-fucosylated.

In yet another aspect, provided herein is an isolated antibody thatbinds a human Siglec-8 and kills mast cells expressing Siglec-8 by ADCCactivity. In some embodiments, the antibody kills mast cells expressingSiglec-8 in vitro (e.g., measured in a cell culture assay as describedin Example 2). In some embodiments, the antibody depletes mast cellsexpressing

Siglec-8 in a subject when a therapeutically effective amount isadministered. In a further embodiment, the antibody depletes at leastabout 20% (e.g. ,at least of the mast cells expressing Siglec-8 in asample obtained from the subject as compared to a baseline level beforetreatment. In any of the embodiments herein, the sample can be a tissuesample or a biological fluid sample. In some embodiments, the tissuesample is one or more selected from the group consisting of: skin, lung,bone marrow, and nasal polyps. In some embodiments, the biological fluidsample is one or more selected from the group consisting of: blood,bronchoalveolar lavage, and nasal lavage. In any of the embodimentsherein, the antibody can be engineered to improve antibody-dependentcell-mediated cytotoxicity (ADCC) activity. In some embodiments, atleast one or two of the heavy chains of the antibody is non-fucosylated.In further embodiments, the antibody may be produced in a cell linehaving a alpha1,6-fucosyltransferase (Fut8) knockout. In some furtherembodiments, the antibody may be produced in a cell line overexpressingβ1,4-N-acetylglycosminyltransferase III (GnT-III). In furtherembodiments, the cell line additionally overexpresses Golgiμ-mannosidase II (ManII). In any of the embodiments herein, the antibodymay comprise at least one amino acid substitution in the Fc region thatimproves ADCC activity. In any of the embodiments herein, the antibodymay be a humanized antibody, a chimeric antibody or a human antibody. Insome embodiments, the antibody is a human IgG1 antibody. In someembodiments, the antibody is a murine antibody. In any of theembodiments herein, the antibody may comprise a heavy chain variableregion comprising (i) HVR-H1 comprising the amino acid sequence of SEQID NO:88, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:91, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:94; and/or a light chain variable region comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:97, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:100, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:103. In a furtherembodiment, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:106; and/or a lightchain variable region comprising the amino acid sequence of SEQ IDNO:109. In any of the embodiments herein, the antibody may comprise aheavy chain variable region comprising a heavy chain variable regioncomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:89, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:92,and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:95;and/or a light chain variable region comprising (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:98, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:101, and (iii) HVR-L3 comprising theamino acid sequence of SEQ ID NO:104. In a further embodiment, theantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO:107; and/or a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:110. In any of theembodiments herein, the antibody may comprise a heavy chain variableregion comprising a heavy chain variable region comprising a heavy chainvariable region comprising (i) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:90, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO:93, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:96; and/or a light chain variable region comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:99, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:102, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:105. In a furtherembodiment, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:108; and/or a lightchain variable region comprising the amino acid sequence of SEQ IDNO:111. In any of the embodiments herein, the antibody may comprise aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the aminoacid sequence of SEQ ID NO:63; and/or wherein the light chain variableregion comprises (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:66. Inany of the embodiments herein, the antibody may comprise a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises (i) HVR-H1 comprising the amino acidsequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequenceselected from SEQ ID NOs:67-70; and/or wherein the light chain variableregion comprises (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:71. Inany of the embodiments herein, the subject can be a human.

In still another aspect, provided herein is an antibody that binds to ahuman Siglec-8 and a non-human primate Siglec-8. In any of theembodiments herein, the antibody may comprise a heavy chain variableregion comprising a heavy chain variable region comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:89, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:92, and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO:95; and/or a light chainvariable region comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:98, (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:101, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:104. In a further embodiment, the antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:107;and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO:110. In any of the embodiments herein, the antibody maycomprise a heavy chain variable region comprising a heavy chain variableregion comprising a heavy chain variable region comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:90, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:93, and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO:96; and/or a light chainvariable region comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:99, (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:102, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:105. In a further embodiment, the antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:108;and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO:111. In any of the embodiments herein, the antibody maybind to an epitope in Domain 1 of human Siglec-8 (e.g., Domain 1 thatcomprises the amino acid sequence of SEQ ID NO:112). In any of theembodiments herein, the antibody may bind to an epitope in Domain 3 ofhuman Siglec-8 (e.g., Domain 3 that comprises the amino acid sequence ofSEQ ID NO:114). In any of the embodiments herein, the antibody may bindto an epitope in Domain 2 of human Siglec-8 (e.g., Domain 2 thatcomprises the amino acid sequence of SEQ ID NO:113). In any of theembodiments herein, the antibody may be a humanized antibody, a chimericantibody or a human antibody. In some embodiments, the antibody is amurine antibody. In some embodiments, the antibody is an IgG1 or IgG4antibody (e.g., human IgG1 or IgG4).

In another aspect, provided herein is an anti-human Siglec 8 antibodythat binds to a fusion protein comprising the amino acid of SEQ IDNO:116 but not to a fusion protein comprising the amino acid of SEQ IDNO:115. In some embodiments, the antibody described herein binds to afusion protein comprising the amino acid of SEQ ID NO:117 but not to afusion protein comprising the amino acid of SEQ ID NO:115. In someembodiments, the antibody described herein binds to a fusion proteincomprising the amino acid of SEQ ID NO:117 but not to a fusion proteincomprising the amino acid of SEQ ID NO:116. In some embodiments herein,the antibody may comprise a heavy chain variable region comprising (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO:88, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:91, and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO:94; and/or a light chainvariable region comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:97, (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:100, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:103. In a further embodiment, the antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:106;and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO:109. In some embodiments herein, the antibody may comprisea heavy chain variable region comprising a heavy chain variable regioncomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:89, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:92,and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:95;and/or a light chain variable region comprising (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:98, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:101, and (iii) HVR-L3 comprising theamino acid sequence of SEQ ID NO:104. In a further embodiment, theantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO:107; and/or a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:110. In some embodimentsherein, the antibody may comprise a heavy chain variable regioncomprising a heavy chain variable region comprising a heavy chainvariable region comprising (i) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:90, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO:93, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:96; and/or a light chain variable region comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:99, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:102, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:105. In a furtherembodiment, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:108; and/or a lightchain variable region comprising the amino acid sequence of SEQ IDNO:111.

In another aspect, provided herein is a humanized antibody that binds toa human Siglec-8, wherein the EC₅₀ in depleting activated humaneosinophils is less than the EC₅₀ of antibody 2E2 or 2C4 to the humanSiglec-8. In some embodiments, the EC₅₀ of the humanized antibody isabout 85% or less than the EC₅₀ of antibody 2E2 or 2C4 to the humanSiglec-8. In some embodiments, the EC₅₀ of the humanized antibody isabout 85%, about 80%, about 70%, about 65%, about 60%, about 55%, about50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%,about 15%, about 10% or about 5% or less than the EC₅₀ of antibody 2E2or 2C4 to the human Siglec-8. In any of the embodiments herein, thehumanized antibody may comprise a heavy chain variable region and alight chain variable region, wherein the heavy chain variable regioncomprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61,(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and/orwherein the light chain variable region comprises (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising theamino acid sequence of SEQ ID NO:66. In any of the embodiments herein,the humanized antibody may comprise a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:6; and/or a light chainvariable region comprising the amino acid sequence selected from SEQ IDNOs:16 or 21. In some embodiments, the humanized antibody comprises aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the aminoacid sequence selected from SEQ ID NOs:67-70; and/or wherein the lightchain variable region comprises (i) HVR-L1 comprising the amino acidsequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequenceof SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence ofSEQ ID NO:71. In any of the embodiments herein, the humanized antibodymay comprise a heavy chain variable region comprising the amino acidsequence selected from SEQ ID NOs:2-14; and/or a light chain variableregion comprising the amino acid sequence selected from SEQ IDNOs:16-24. In any of the embodiments herein, the antibody may comprise aheavy chain variable region comprising (i) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:88, (ii) HVR-H2 comprising the amino acidsequence of SEQ ID NO:91, and (iii) HVR-H3 comprising the amino acidsequence of SEQ ID NO:94; and/or a light chain variable regioncomprising (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:97, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:100,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:103. Inany of the embodiments herein, the antibody may comprise a heavy chainvariable region comprising a heavy chain variable region comprising (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO:89, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:92, and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO:95; and/or a light chainvariable region comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:98, (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:101, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:104. In any of the embodiments herein, the antibody may comprise aheavy chain variable region comprising a heavy chain variable regioncomprising a heavy chain variable region comprising (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:90, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:93, and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO:96; and/or a light chainvariable region comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:99, (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:102, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:105. In any of the embodiments herein, the antibody may comprise aheavy chain Fc region comprising a human IgG Fc region. In furtherembodiments, the human IgG Fc region comprises a human IgG1 or IgG4. Insome embodiments, the human IgG1 comprises the amino acid sequence ofSEQ ID NO:78. In some embodiments, the human IgG4 comprises the aminoacid sequence of SEQ ID NO:79. In any of the embodiments herein, theantibody may comprise a heavy chain comprising the amino acid sequenceof SEQ ID NO:75; and/or a light chain comprising the amino acid sequenceselected from SEQ ID NOs:76 or 77. In some embodiments, the human IgG4comprises the amino acid substitution S228P, wherein the amino acidresidues are numbered according to the EU index as in Kabat. In any ofthe embodiments herein, the antibody may comprise a heavy chaincomprising the amino acid sequence of SEQ ID NO:87; and/or a light chaincomprising the amino acid sequence of SEQ ID NO:76.

In another aspect, provided herein is a nucleic acid encoding anyantibody described above and herein. In yet another aspect, providedherein is a vector comprising a nucleic acid described herein. In oneembodiment, the vector is an expression vector. In yet another aspect,provided herein is a host cell comprising a nucleic acid describedherein. In some embodiments, the host cell expresses and produces theantibody.

In another aspect, provided herein is a method of producing an antibodycomprising culturing a host cell comprising one or more nucleic acidsencoding an antibody described herein under a condition that producesthe antibody. In some embodiments, the method further comprisesrecovering the antibody produced by the host cell. Also provided hereinis an anti-Siglec-8 antibody produced by the method. Also providedherein is an antigen-binding fragment of an anti-Siglec-8 antibodydescribed herein.

In another aspect, provided herein is a pharmaceutical compositioncomprising an antibody described above and herein or an antigen-bindingfragment thereof and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a composition comprising anantibody or fragment thereof that specifically binds to human Siglec-8,wherein the antibody comprises a Fc region and N-glycoside-linkedcarbohydrate chains linked to the Fc region, wherein less than 50% ofthe N-glycoside-linked carbohydrate chains in the composition contain afucose residue. In some embodiments, substantially none of theN-glycoside-linked carbohydrate chains contain a fucose residue. In someembodiments, the antibody is a humanized antibody, a chimeric antibodyor a human antibody. In some embodiments, the antibody comprises a heavychain variable region and a light chain variable region, wherein theheavy chain variable region comprises (i) HVR-H1 comprising the aminoacid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acidsequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acidsequence of SEQ ID NO:63; and/or wherein the light chain variable regioncomprises (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:64,(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65, and(iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:66. In someembodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NOs:2-10; and/or a lightchain variable region comprising the amino acid sequence of SEQ IDNOs:16-22. In some embodiments, the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises (i) HVR-H1 comprising the amino acidsequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequenceselected from SEQ ID NOs:67-70; and/or wherein the light chain variableregion comprises (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:71. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence selected from SEQ ID NOs:11-14;and/or a light chain variable region comprising the amino acid sequenceselected from SEQ ID NOs:23-24. In some embodiments, the antibodycomprises a heavy chain variable region comprising the amino acidsequence selected from SEQ ID NOs:2-14; and/or a light chain variableregion comprising the amino acid sequence selected from SEQ IDNOs:16-24. In any of the embodiments herein, the composition may furthercomprise a pharmaceutically acceptable carrier. In any of theembodiments herein, the binding affinity and/or binding avidity of theantibody to a human Siglec-8 can be higher than the binding affinityand/or binding avidity of antibody 2E2 or 2C4 to the human Siglec-8. Inany of the embodiments herein, the antibody may have a Tm of at leastabout 70° C. to at least about 72° C. in a thermal shift assay. In someembodiments, the antibody has a Tm of about 70° C., about 71° C., orabout 72° C. In some embodiments, the antibody has the same or higher Tmas compared to a chimeric 2C4 antibody. In some embodiments, theantibody has the same or higher Tm as compared to an antibody having aheavy chain comprising the amino acid sequence of SEQ ID NO:84 and alight chain comprising the amino acid sequence of SEQ ID NO:85.

In another aspect, provided herein is a method of treating or preventinga disease mediated by cells expressing Siglec-8 in a subject, the methodcomprising administering to the subject an effective amount of anantibody described herein or an antigen-binding fragment thereof or acomposition described herein. In some embodiments, the disease is aneosinophil mediated-disease. In some embodiments, the disease is a mastcell mediated-disease. In some embodiments, the disease is selected fromthe group consisting of asthma, allergic rhinitis, nasal polyposis,atopic dermatitis, chronic urticaria, mastocytosis, eosinophilicleukemia, and hypereosinophilic syndrome. In some embodiments, thedisease is selected from the group consisting of pauci granulocyticasthma, acute or chronic airway hypersensitivity, eosinophilicesophagitis, Churg-Strauss syndrome, inflammation associated with acytokine, inflammation associated with cells expressing Siglec-8,malignancy associated with cells expressing Siglec-8, physicalurticaria, cold urticaria, pressure-urticaria, bullous pemphigoid, foodallergy, and allergic bronchopulmonary aspergillosis (ABPA). In someembodiments, the antibody inhibits one or more symptoms of an allergicreaction. In some embodiments, the allergic reaction is a Type Ihypersensitivity reaction. In any of the embodiments herein, the subjectmay be suffering from asthma that is not adequately controlled by aninhaled corticosteroid, a short acting β2 agonist, a long acting β2agonist, or a combination thereof.

In another aspect, provided herein is a method of depleting mast cellsexpressing Siglec-8 in a subject comprising administering to the subjectan effective amount of an antibody that binds to human Siglec-8, whereinthe antibody kills mast cells expressing Siglec-8 by ADCC activity. Insome embodiments, the antibody kills mast cells expressing Siglec-8 invitro (e.g., measured in a cell culture assay as described in Example2). In some embodiments, the antibody depletes at least about 20% of themast cells expressing Siglec-8 in a sample obtained from the subject ascompared to a baseline level before treatment. In any of the embodimentsherein, the sample can be a tissue sample or a biological fluid sample.In some embodiments, the tissue sample is one or more selected from thegroup consisting of: skin, lung, bone marrow, and nasal polyps. In someembodiments, the biological fluid sample is one or more selected fromthe group consisting of: blood, bronchoalveolar lavage, and nasallavage. In any of the embodiments herein, the antibody can be engineeredto improve antibody-dependent cell-mediated cytotoxicity (ADCC)activity. In any of the embodiments herein, the antibody may comprisetwo heavy chains and wherein at least one of the two or both heavychains of the antibody is non-fucosylated. In further embodiments, theantibody may be produced in a cell line having aalpha1,6-fucosyltransferase (Fut8) knockout. In some furtherembodiments, the antibody may be produced in a cell line overexpressingβ1,4-N-acetylglycosminyltransferase III (GnT-III). In furtherembodiments, the cell line additionally overexpresses Golgiμ-mannosidase II (ManII). In any of the embodiments herein, the antibodymay comprise at least one amino acid substitution in the Fc region thatimproves ADCC activity. In any of the embodiments herein, the antibodymay be a humanized antibody, a chimeric antibody or a human antibody. Insome embodiments, the antibody is a human IgG1 antibody. In any of theembodiments herein, the antibody may comprise a heavy chain variableregion comprising (i) HVR-H1 comprising the amino acid sequence of SEQID NO:88, (ii) HVR-H2 comprising the amino acid sequence of SEQ IDNO:91, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:94; and/or a light chain variable region comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:97, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:100, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:103. In a furtherembodiment, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:106; and/or a lightchain variable region comprising the amino acid sequence of SEQ IDNO:109. In any of the embodiments herein, the antibody may comprise aheavy chain variable region comprising a heavy chain variable regioncomprising (i) HVR-H1 comprising the amino acid sequence of SEQ IDNO:89, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:92,and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:95;and/or a light chain variable region comprising (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:98, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:101, and (iii) HVR-L3 comprising theamino acid sequence of SEQ ID NO:104. In a further embodiment, theantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO:107; and/or a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:110. In any of theembodiments herein, the antibody may comprise a heavy chain variableregion comprising a heavy chain variable region comprising a heavy chainvariable region comprising (i) HVR-H1 comprising the amino acid sequenceof SEQ ID NO:90, (ii) HVR-H2 comprising the amino acid sequence of SEQID NO:93, and (iii) HVR-H3 comprising the amino acid sequence of SEQ IDNO:96; and/or a light chain variable region comprising (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:99, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:102, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:105. In a furtherembodiment, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:108; and/or a lightchain variable region comprising the amino acid sequence of SEQ IDNO:111. In any of the embodiments herein, the antibody may comprise aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the aminoacid sequence of SEQ ID NO:63; and/or wherein the light chain variableregion comprises (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:66. Inany of the embodiments herein, the antibody may comprise a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises (i) HVR-H1 comprising the amino acidsequence of SEQ ID NO:61, (ii) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:62, and (iii) HVR-H3 comprising the amino acid sequenceselected from SEQ ID NOs:67-70; and/or wherein the light chain variableregion comprises (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:71. Inany of the embodiments herein, the subject has a disease mediated bycells expressing Siglec-8. In some embodiments, the disease is selectedfrom the group consisting of asthma, allergic rhinitis, nasal polyposis,atopic dermatitis, chronic urticaria, mastocytosis, eosinophilicleukemia, and hypereosinophilic syndrome. In some embodiments, thedisease is selected from the group consisting of pauci granulocyticasthma, acute or chronic airway hypersensitivity, eosinophilicesophagitis, Churg-Strauss syndrome, inflammation associated with acytokine, inflammation associated with cells expressing Siglec-8,malignancy associated with cells expressing Siglec-8, physicalurticaria, cold urticaria, pressure-urticaria, bullous pemphigoid, foodallergy, and allergic bronchopulmonary aspergillosis (ABPA). In someembodiments, the antibody inhibits one or more symptoms of an allergicreaction. In some embodiments, the allergic reaction is a Type Ihypersensitivity reaction. In any of the embodiments herein, the subjectmay be suffering from asthma that is not adequately controlled by aninhaled corticosteroid, a short acting β2 agonist, a long acting β2agonist, or a combination thereof. In any of the embodiments herein, thesubject can be a human.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequence alignment showing the comparison of heavy chainsequences of humanized antibodies. Framework residues within 4 Å of theCDRs in the molecular model of mouse 2E2 antibody are represented by a*; residues within 4 Å of the CDRs and VCI residues that were differentin the acceptor human framework are highlighted in {circumflex over( )}, back-mutations of these residues in the acceptor human frameworkto mouse are indicated by @; Somatic mutations from human germline areindicated by ^(o); if those residues were different from the AF471521 FWand mou2E2 (i.e., mouse 2E2) or different from the AF471521 frameworkand identical to the mou2E2 (i.e., mouse 2E2) the corresponding residuesin the acceptor human framework were back-mutated to human germline andare indicated by +. Sequences highlighted in version 2 of the heavychain represent a straight graft of mou2E2 (i.e., mouse 2E2) CDRs intothe closest human germline family (i.e., closest germline sequence).m2E2 VH corresponds to SEQ ID NO:1; Human FW AF471521 corresponds to SEQID NO:120; Germline X92218 VH366 corresponds to SEQ ID NO:121; 2E2 RHAcorresponds to SEQ ID NO:2; 2E2 RHB corresponds to SEQ ID NO:3; 2E2 RHCcorresponds to SEQ ID NO:4; 2E2 RHD corresponds to SEQ ID NO:5; 2E2 RHEcorresponds to SEQ ID NO:6; 2E2 RHF corresponds to SEQ ID NO:7; 2E2 RHGcorresponds to SEQ ID NO:8; IGHV4-59 FW corresponds to SEQ ID NO:122;2E2 RHA2 corresponds to SEQ ID NO:9; and 2E2 RHB2 corresponds to SEQ IDNO:10.

FIG. 2 is a sequence alignment showing the comparison of light chainsequences of humanized antibodies. Framework residues within 4 Å of theCDRs in the molecular model of mouse 2E2 antibody are represented by a*; residues within 4 Å of the CDRs and VCI residues that are differentin the acceptor human framework are indicated by +; back-mutations ofthese residues in the human FW to mouse are indicated by @;back-mutation to human germline of residues which in the X93721framework are different from the mouse are indicated by #. m2E2 VKcorresponds to SEQ ID NO:15; X93721 corresponds to SEQ ID NO:123;Germline X01668 corresponds to SEQ ID NO:124; m2E2 RKA corresponds toSEQ ID NO:16; m2E2 RKB corresponds to SEQ ID NO:17; m2E2 RKC correspondsto SEQ ID NO:18; m2E2 RKD corresponds to SEQ ID NO:19; m2E2 RKEcorresponds to SEQ ID NO:20; m2E2 RKF corresponds to SEQ ID NO:21; andm2E2 RKG corresponds to SEQ ID NO:22.

FIG. 3 is a sequence alignment showing the comparison of CDR sequencesthat were mutated in both light and heavy chains of humanizedantibodies. CDR residues that were mutated in the variant to be closerto the germline sequence are indicated by #. 2E2 RKA corresponds to SEQID NO:16; 2E2 RKF corresponds to SEQ ID NO:21; 2E2 RKA F-Y mutcorresponds to SEQ ID NO:23; 2E2 RKF F-Y mut corresponds to SEQ IDNO:24; 2E2 RHA corresponds to SEQ ID NO:2; 2E2 RHE corresponds to SEQ IDNO:6; 2E2 RHE S-G mut corresponds to SEQ ID NO:11; 2E2 RHE E-D mutcorresponds to SEQ ID NO:12; 2E2 RHE Y-V mut corresponds to SEQ IDNO:13; and 2E2 RHE E-D triple mut corresponds to SEQ ID NO:14.

FIG. 4 is a graph showing a comparison of Siglec-8 antigen binding bypurified candidate antibodies encoded by chimeric 2C4 or combinations of2E2 RHE with 2E2 RKA, 2E2 RKF, 2E2 RKA CDR3 mutant, or 2E2 RKF CDR3mutant, heated for 10 min at the indicated temperature, then cooled to4° C. before performing the Siglec-8 binding ELISA.

FIG. 5 is a graph showing the Tm of purified 2C4 candidate antibodies ascompared to ch2C4 (i.e., chimeric 2C4 antibody).

FIG. 6 is a graph showing the stability of chimeric and humanizedanti-Siglec-8 antibodies after being frozen at −20° C. for 60 minutesand thawed at room temperature.

FIG. 7 is a graph showing killing of eosinophils with anti-Siglec-8antibodies. Total peripheral blood leukocytes were incubated in thepresence of the indicated anti-Siglec-8 and control antibodiesconcentrations for 16 hours. Reduction of eosinophil numbers weremonitored by flow cytometry and quantified as a loss of CD16-negativeIL5Rα+ cells with high side-scatter (SSC^(HI)). p FIGS. 8A & 8B are aseries of graphs showing in vitro apoptosis and in vivo depletion ofhuman mast cells by anti-Siglec-8 antibodies. FIG. 8A. NK-cell mediatedantibody-dependent cell-mediated cytotoxicity (ADCC) activity of HEKAIgG4 antibody, non-fucosylated HEKA IgG1 antibody and low fucosechimeric 1H10 IgG1 antibodies on primary human mast cells fromperitoneal lavage of NSGS mice engrafted with human hematopoietic stemcells as demonstrated by LDH release at 48 hours. Control indicates afucosylated human IgG1 isotype antibody that does not bind to Siglec-8.FIG. 8B. Depletion of Siglec-8 positive mast cells in vivo. Siglec-8transgenic mice selectively expressing Siglec-8 on the surface of mastcells at levels comparable to those on human mast cells were treatedintraperotineally with HEKA IgG4 antibody, non-fucosylated HEKA IgG1antibody, murine 1C3 antibody, or a human IgG4 isotype control antibodythat does not bind to Siglec-8. n=4 mice per group.

FIG. 9 is a graph showing inhibition of a Type I hypersensitivityreaction in humanized mice by a humanized anti-Siglec-8 IgG4 antibody.Passive cutaneous anaphylaxis response was induced in the ears ofengrafted NSGS mice by sensitization with anti-NP-IgE (delivered to theright ear) or PBS control (delivered to the left ear) and NP-BSAadministered 24 hours later sensitization. Mice were treated with HEKAIgG4 or a human IgG4 isotype control antibody that does not bind toSiglec-8 either 24 hours pre-sensitization as indicated by * or 2 hourspost-sensitization as indicated by @. Mean change in ear thickness andstandard errors at 3 hours or 24 hours post-challenge are shown. n=5mice per group for HEKA IgG4 antibody treated mice and n=4 mice pergroup for isotype control antibody treated mice.

FIG. 10 is a series of histograms showing binding of murineanti-Siglec-8 monoclonal antibodies 2E2, 1C3, and 1H10 to baboon andhuman eosinophils (CD49⁺ CD16⁻ SSC^(high) cells) as demonstrated by flowcytometry. Histograms show number of baboon or human blood eosinophilsplotted against fluorescence intensity for each antibody in comparisonto a mouse IgG1 isotype control antibody that does not bind to Siglec-8.

DETAILED DESCRIPTION I. Definitions.

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. As used in thisspecification and the appended claims, the singular forms “a”, “an” and“the” include plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to “a molecule” optionallyincludes a combination of two or more such molecules, and the like.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

The term “antibody” includes polyclonal antibodies, monoclonalantibodies (including full length antibodies which have animmunoglobulin Fc region), antibody compositions with polyepitopicspecificity, multispecific antibodies (e.g., bispecific antibodies,diabodies, and single-chain molecules, as well as antibody fragments(e.g., Fab, F(ab′)₂, and Fv). The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (CH),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, havingheavy chains designated α, δ, ε, γ and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in the CH sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. IgG1antibodies can exist in multiple polymorphic variants termed allotypes(reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any ofwhich are suitable for use in the invention. Common allotypic variantsin human populations are those designated by the letters a,f,n,z.

An “isolated” antibody is one that has been identified, separated and/orrecovered from a component of its production environment (e.g.,naturally or recombinantly). In some embodiments, the isolatedpolypeptide is free of association with all other components from itsproduction environment. Contaminant components of its productionenvironment, such as that resulting from recombinant transfected cells,are materials that would typically interfere with research, diagnosticor therapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or non-proteinaceous solutes. In someembodiments, the polypeptide is purified: (1) to greater than 95% byweight of antibody as determined by, for example, the Lowry method, andin some embodiments, to greater than 99% by weight; (1) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under non-reducing or reducing conditions usingCoomassie blue or silver stain. Isolated antibody includes the antibodyin situ within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,an isolated polypeptide or antibody is prepared by at least onepurification step.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. In some embodiments, monoclonalantibodies have a C-terminal cleavage at the heavy chain and/or lightchain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved atthe C-terminus of heavy chain and/or light chain. In some embodiments,the C-terminal cleavage removes a C-terminal lysine from the heavychain. In some embodiments, monoclonal antibodies have an N-terminalcleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4,or 5 amino acid residues are cleaved at the N-terminus of heavy chainand/or light chain. In some embodiments truncated forms of monoclonalantibodies can be made by recombinant techniques. In some embodiments,monoclonal antibodies are highly specific, being directed against asingle antigenic site. In some embodiments, monoclonal antibodies arehighly specific, being directed against multiple antigenic sites (suchas a bispecific antibody or a multispecific antibody). The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method, recombinant DNA methods,phage-display technologies, and technologies for producing human orhuman-like antibodies in animals that have parts or all of the humanimmunoglobulin loci or genes encoding human immunoglobulin sequences.

The term “naked antibody” refers to an antibody that is not conjugatedto a cytotoxic moiety or radiolabel.

The terms “full-length antibody,” “intact antibody” or “whole antibody”are used interchangeably to refer to an antibody in its substantiallyintact form, as opposed to an antibody fragment. Specifically wholeantibodies include those with heavy and light chains including an Fcregion. The constant domains may be native sequence constant domains(e.g., human native sequence constant domains) or amino acid sequencevariants thereof In some cases, the intact antibody may have one or moreeffector functions.

An “antibody fragment” comprises a portion of an intact antibody, theantigen binding and/or the variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂ and Fvfragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

Papain digestion of antibodies produced two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H)1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdifferent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having a fewadditional residues at the carboxy terminus of the C_(H)1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the VH and VL antibody domains connected into asingle polypeptide chain. In some embodiments, the sFv polypeptidefurther comprises a polypeptide linker between the V_(H) and V_(L)domains which enables the sFv to form the desired structure for antigenbinding. For a review of the sFv, see Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,Springer-Verlag, New York, pp. 269-315 (1994).

“Functional fragments” of the antibodies of the invention comprise aportion of an intact antibody, generally including the antigen bindingor variable region of the intact antibody or the Fv region of anantibody which retains or has modified FcR binding capability. Examplesof antibody fragments include linear antibody, single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is (are) identical with or homologous to correspondingsequences in antibodies derived from another species or belonging toanother antibody class or subclass, as well as fragments of suchantibodies, so long as they exhibit the desired biological activity(U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). Chimeric antibodies of interest herein includePRIMATIZED® antibodies wherein the antigen-binding region of theantibody is derived from an antibody produced by, e.g., immunizingmacaque monkeys with an antigen of interest. As used herein, “humanizedantibody” is used as a subset of “chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR of therecipient are replaced by residues from an HVR of a non-human species(donor antibody) such as mouse, rat, rabbit or non-human primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance, such as binding affinity. In general, a humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableloops correspond to those of a non-human immunoglobulin sequence, andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence, although the FR regions may include one or moreindividual FR residue substitutions that improve antibody performance,such as binding affinity, isomerization, immunogenicity, etc. In someembodiments, the number of these amino acid substitutions in the FR areno more than 6 in the H chain, and in the L chain, no more than 3. Thehumanized antibody optionally will also comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, for example,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409. In some embodiments, humanized antibodies are directedagainst a single antigenic site. In some embodiments, humanizedantibodies are directed against multiple antigenic sites. An alternativehumanization method is described in U.S. Pat. No. 7,981,843 and U.S.Patent Application Publication No. 2006/0134098.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy chain and light chain may be referred toas “VH” and “VL”, respectively. These domains are generally the mostvariable parts of the antibody (relative to other antibodies of the sameclass) and contain the antigen binding sites.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody-variable domain that are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003)). Indeed, naturallyoccurring camelid antibodies consisting of a heavy chain only arefunctional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheHVRs that are Kabat complementarity-determining regions (CDRs) are basedon sequence variability and are the most commonly used (Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institute of Health, Bethesda, Md. (1991)).Chothia HVRs refer instead to the location of the structural loops(Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat Chothia Contact L1 L24-L34 L26-L34 L30-L36 L2 L50-L56 L50-L56L46-L55 L3 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H32 H30-H35B (KabatNumbering) H1 H31-H35 H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65H53-H56 H47-H58 H3 H95-H102 H95-H102 H93-H101

Unless otherwise indicated, the variable-domain residues (HVR residuesand framework region residues) are numbered according to Kabat et al.,supra.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a VL or VH framework derived froma human immunoglobulin framework or a human consensus framework. Anacceptor human framework “derived from” a human immunoglobulin frameworkor a human consensus framework may comprise the same amino acid sequencethereof, or it may contain pre-existing amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For example, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence in that program's alignment of A and B, and where Y isthe total number of amino acid residues in B. It will be appreciatedthat where the length of amino acid sequence A is not equal to thelength of amino acid sequence B, the % amino acid sequence identity of Ato B will not equal the % amino acid sequence identity of B to A.

An antibody that “binds to”, “specifically binds to” or is “specificfor” a particular a polypeptide or an epitope on a particularpolypeptide is one that binds to that particular polypeptide or epitopeon a particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope. In some embodiments, binding of ananti-Siglec-8 antibody described herein to an unrelated, non-Siglec-8polypeptide is less than about 10% of the antibody binding to Siglec-8as measured by methods known in the art (e.g., enzyme-linkedimmunosorbent assay (ELISA)). In some embodiments, the antibody thatbinds to a Siglec-8 (e.g., Siglec-8 Fc fusion protein in dimer form (SEQID NO:74)) has a dissociation constant (1(d) of ≤1 μM, ≤100 nM, ≤10 nM,≤2 nM, ≤1 nM, ≤0.7 nM, ≤0.6 nM, ≤0.5 nM, <0.1 nM, ≤0.01 nM, or ≤0.001 nM(e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to10⁻¹³ M).

The term “Siglec-8” as used herein refers to a human Siglec-8 protein.The term also includes naturally occurring variants of Siglec-8,including splice variants or allelic variants. The amino acid sequenceof an exemplary human Siglec-8 is shown in SEQ ID NO:72. The amino acidsequence of another exemplary human Siglec-8 is shown in SEQ ID NO:73.In some embodiments, a human Siglec-8 protein comprises the humanSiglec-8 extracellular domain fused to an immunoglobulin Fc region. Theamino acid sequence of an exemplary human Siglec-8 extracellular domainfused to an immunoglobulin Fc region is shown in SEQ ID NO:74. The aminoacid sequence underlined in SEQ ID NO:74 indicates the Fc region of theSiglec-8 Fc fusion protein amino acid sequence.

Human Siglec-8 Amino Acid Sequence

(SEQ ID NO: 72) GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGAGATALAFLSFCIIFIIVRSCRKKSARPAAGVGDTGMEDAKAIRGSASQGPLTESWKDGNPLKKPPPAVAPSSGEEGELHYATLSFHKVKPQDPQGQEATDSEYSEIKIHKRETAETQACLRNHNPSSKEVRG

Human Siglec-8 Amino Acid Sequence

(SEQ ID NO: 73) GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHPRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGAGATALAFLSFCIIFIIVRSCRKKSARPAAGVGDTGMEDAKAIRGSASQGPLTESWKDGNPLKKPPPAVAPSSGEEGELHYATLSFHKVKPQDPQGQEATDSEYSEIKIHKRETAETQACLRNHNPSSKEVRG

Siglec-8 Fc Fusion Protein Amino Acid Sequence

(SEQ ID NO: 74) GYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Antibodies that “induce apoptosis” or are “apoptotic” are those thatinduce programmed cell death as determined by standard apoptosis assays,such as binding of annexin V, fragmentation of DNA, cell shrinkage,dilation of endoplasmic reticulum, cell fragmentation, and/or formationof membrane vesicles (called apoptotic bodies). For example, theapoptotic activity of the anti-Siglec-8 antibodies of the presentinvention can be showed by staining cells with annexin V.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and Bcell activation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., natural killer (NK) cells,neutrophils and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are required for killing of the target cell by this mechanism.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. Fc expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9: 457-92 (1991). In some embodiments, ananti-Siglec-8 antibody described herein enhances ADCC. To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. Nos. 5,500,362 or 5,821,337 may be performed.Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and natural killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as that disclosed inClynes et al., PNAS USA 95:652-656 (1998). Other Fc variants that alterADCC activity and other antibody properties include those disclosed byGhetie et al., Nat Biotech. 15:637-40, 1997; Duncan et al, Nature332:563-564, 1988; Lund et al., J. Immunol 147:2657-2662, 1991; Lund etal, Mol Immunol 29:53-59, 1992; Alegre et al, Transplantation57:1537-1543, 1994; Hutchins et al., Proc Natl. Acad Sci USA92:11980-11984, 1995; Jefferis et al, Immunol Lett. 44:111-117, 1995;Lund et al., FASEB J 9:115-119, 1995; Jefferis et al, Immunol Lett54:101-104, 1996; Lund et al, J Immunol 157:4963-4969, 1996; Armour etal., Eur J Immunol 29:2613-2624, 1999; Idusogie et al, J Immunol164:4178-4184, 200; Reddy et al, J Immunol 164:1925-1933, 2000; Xu etal., Cell Immunol 200:16-26, 2000; Idusogie et al, J Immunol166:2571-2575, 2001; Shields et al., J Biol Chem 276:6591-6604, 2001;Jefferis et al, Immunol Lett 82:57-65. 2002; Presta et al., Biochem SocTrans 30:487-490, 2002; Lazar et al., Proc. Natl. Acad. Sci. USA103:4005-4010, 2006; U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,194,551; 6,737,056; 6,821,505; 6,277,375; 7,335,742; and 7,317,091.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof.Suitable native-sequence Fc regions for use in the antibodies of theinvention include human IgG1, IgG2, IgG3 and IgG4. A single amino acidsubstitution (S228P according to Kabat numbering; designated IgG4Pro)may be introduced to abolish the heterogeneity observed in recombinantIgG4 antibody. See Angal, S. et al. (1993) Mol Immunol 30, 105-108.

“Non-fucosylated” or “fucose-deficient” antibody refers to aglycosylation antibody variant comprising an Fc region wherein acarbohydrate structure attached to the Fc region has reduced fucose orlacks fucose. In some embodiments, an antibody with reduced fucose orlacking fucose has improved ADCC function. Non-fucosylated orfucose-deficient antibodies have reduced fucose relative to the amountof fucose on the same antibody produced in a cell line. Anon-fucosylated or fucose-deficient antibody composition contemplatedherein is a composition wherein less than about 50% of the N-linkedglycans attached to the Fc region of the antibodies in the compositioncomprise fucose.

The terms “fucosylation”or “fucosylated” refers to the presence offucose residues within the oligosaccharides attached to the peptidebackbone of an antibody. Specifically, a fucosylated antibody comprisesα (1,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc)residue in one or both of the N-linked oligosaccharides attached to theantibody Fc region, e.g. at position Asn 297 of the human IgG1 Fc domain(EU numbering of Fc region residues). Asn297 may also be located about+3 amino acids upstream or downstream of position 297, i.e. betweenpositions 294 and 300, due to minor sequence variations inimmunoglobulins.

The “degree of fucosylation” is the percentage of fucosylatedoligosaccharides relative to all oligosaccharides identified by methodsknown in the art e.g., in an N-glycosidase F treated antibodycomposition assessed by matrix-assisted laser desorption-ionizationtime-of-flight mass spectrometry (MALDI TOF MS). In a composition of a“fully fucosylated antibody” essentially all oligosaccharides comprisefucose residues, i.e. are fucosylated. In some embodiments, acomposition of a fully fucosylated antibody has a degree of fucosylationof at least about 90%. Accordingly, an individual antibody in such acomposition typically comprises fucose residues in each of the twoN-linked oligosaccharides in the Fc region. Conversely, in a compositionof a “fully non-fucosylated” antibody essentially none of theoligosaccharides are fucosylated, and an individual antibody in such acomposition does not contain fucose residues in either of the twoN-linked oligosaccharides in the Fc region. In some embodiments, acomposition of a fully non-fucosylated antibody has a degree offucosylation of less than about 10%. In a composition of a “partiallyfucosylated antibody” only part of the oligosaccharides comprise fucose.An individual antibody in such a composition can comprise fucoseresidues in none, one or both of the N-linked oligosaccharides in the Fcregion, provided that the composition does not comprise essentially allindividual antibodies that lack fucose residues in the N-linkedoligosaccharides in the Fc region, nor essentially all individualantibodies that contain fucose residues in both of the N-linkedoligosaccharides in the Fc region. In one embodiment, a composition of apartially fucosylated antibody has a degree of fucosylation of about 10%to about 80% (e.g., about 50% to about 80%, about 60% to about 80%, orabout 70% to about 80%).

“Binding affinity” as used herein refers to the strength of thenon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). In someembodiments, the affinity of an antibody for a Siglec-8 (which may be adimer, such as the Siglec-8-Fc fusion protein described herein) cangenerally be represented by a dissociation constant (Kd). Affinity canbe measured by common methods known in the art, including thosedescribed herein.

“Binding avidity” as used herein refers to the binding strength ofmultiple binding sites of a molecule (e.g., an antibody) and its bindingpartner (e.g., an antigen).

An “isolated” nucleic acid molecule encoding the antibodies herein is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the environment in which it was produced. In some embodiments, theisolated nucleic acid is free of association with all componentsassociated with the production environment. The isolated nucleic acidmolecules encoding the polypeptides and antibodies herein is in a formother than in the form or setting in which it is found in nature.Isolated nucleic acid molecules therefore are distinguished from nucleicacid encoding the polypeptides and antibodies herein existing naturallyin cells.

The term “pharmaceutical formulation” refers to a preparation that is insuch form as to permit the biological activity of the active ingredientto be effective, and that contains no additional components that areunacceptably toxic to a subject to which the formulation would beadministered. Such formulations are sterile.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. An individual is successfully “treated”, for example, if oneor more symptoms associated with a disorder (e.g., aneosinophil-mediated disease) are mitigated or eliminated. For example,an individual is successfully “treated” if treatment results inincreasing the quality of life of those suffering from a disease,decreasing the dose of other medications required for treating thedisease, reducing the frequency of recurrence of the disease, lesseningseverity of the disease, delaying the development or progression of thedisease, and/or prolonging survival of individuals.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the individual.

As used herein, the term “prevention” includes providing prophylaxiswith respect to occurrence or recurrence of a disease in an individual.An individual may be predisposed to, susceptible to a disorder, or atrisk of developing a disorder, but has not yet been diagnosed with thedisorder. In some embodiments, anti-Siglec-8 antibodies described hereinare used to delay development of a disorder.

As used herein, an individual “at risk” of developing a disorder may ormay not have detectable disease or symptoms of disease, and may or maynot have displayed detectable disease or symptoms of disease prior tothe treatment methods described herein. “At risk” denotes that anindividual has one or more risk factors, which are measurable parametersthat correlate with development of the eosinophil-mediated disorderand/or mast cell mediated, as known in the art. An individual having oneor more of these risk factors has a higher probability of developing thedisorder than an individual without one or more of these risk factors.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired or indicatedeffect, including a therapeutic or prophylactic result. An effectiveamount can be provided in one or more administrations. A“therapeutically effective amount” is at least the minimum concentrationrequired to effect a measurable improvement of a particular disorder. Atherapeutically effective amount herein may vary according to factorssuch as the disease state, age, sex, and weight of the patient, and theability of the antibody to elicit a desired response in the individual.A therapeutically effective amount may also be one in which any toxic ordetrimental effects of the antibody are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at the dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at the earlier stage of disease, the prophylactically effectiveamount can be less than the therapeutically effective amount.

“Chronic” administration refers to administration of the medicament(s)in a continuous as opposed to acute mode, so as to main the initialtherapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

As used herein, an “individual” or a “subject” is a mammal. A “mammal”for purposes of treatment includes humans, domestic and farm animals,and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle,pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In someembodiments, the individual or subject is human.

II. Anti-Siglec-8 Antibodies and Compositions

In one aspect, the invention provides isolated antibodies that bind to ahuman Siglec-8. In some embodiments, an anti-Siglec-8 antibody describedherein has one or more of the following characteristics: (1) binds ahuman Siglec-8; (2) binds to an extracellular domain of a humanSiglec-8; (3) binds a human Siglec-8 with a higher affinity than mouseantibody 2E2 and/or mouse antibody 2C4; (4) binds a human Siglec-8 witha higher avidity than mouse antibody 2E2 and/or mouse antibody 2C4; (5)has a T_(m) of about 70° C.-72° C. or higher in a thermal shift assay;(6) with a reduced degree of fucosylation or is non-fucosylated; (7)binds a human Siglec-8 expressed on eosinophils and induces apoptosis ofeosinophils; (8) binds a human Siglec-8 expressed on mast cells anddepletes mast cells; (9) binds a human Siglec-8 expressed on mast cellsand inhibits FcεRI-dependent activities of mast cells (e.g., histaminerelease, PGD₂ release, Ca²⁺ flux, and/or β-hexosaminidase release,etc.); (10) has been engineered to improve ADCC activity; (11) binds ahuman Siglec-8 expressed on mast cells and kills mast cells by ADCCactivity (in vitro, and/or in vivo); (12) binds to Siglec-8 of a humanand a non-human primate; (13) binds to Domain 1, Domain 2, and/or Domain3 of human Siglec-8, or binds a Siglec-8 polypeptide comprising Domain1, Domain 2, and/or Domain 3 of human Siglec-8 (e.g., fusion proteinsdescribed herein); and (14) depletes activated eosinophils with an EC₅₀less than the EC₅₀ of mouse antibody 2E2 or 2C4.

In one aspect, the invention provides antibodies that bind to a humanSiglec-8. In some embodiments, the human Siglec-8 comprises an aminoacid sequence of SEQ ID NO:72. In some embodiments, the human Siglec-8comprises an amino acid sequence of SEQ ID NO:73. In some embodiments,the antibody described herein binds to an epitope in Domain 1 of humanSiglec-8, wherein Domain 1 comprises the amino acid sequence of SEQ IDNO: 112. In some embodiments, the antibody described herein binds to anepitope in Domain 2 of human Siglec-8, wherein Domain 2 comprises theamino acid sequence of SEQ ID NO: 113. In some embodiments, the antibodydescribed herein binds to an epitope in Domain 3 of human Siglec-8,wherein Domain 3 comprises the amino acid sequence of SEQ ID NO: 114. Insome embodiments, the antibody described herein binds to a fusionprotein comprising the amino acid of SEQ ID NO:116 but not to a fusionprotein comprising the amino acid of SEQ ID NO:115. In some embodiments,the antibody described herein binds to a fusion protein comprising theamino acid of SEQ ID NO:117 but not to a fusion protein comprising theamino acid of SEQ ID NO:115. In some embodiments, the antibody describedherein binds to a fusion protein comprising the amino acid of SEQ IDNO:117 but not to a fusion protein comprising the amino acid of SEQ IDNO:116. In some embodiments, the antibody described herein binds to alinear epitope in the extracellular domain of human Siglec-8. In someembodiments, the antibody described herein binds to a conformationalepitope in the extracellular domain of human Siglec-8. In someembodiments, an antibody described herein binds to a human Siglec-8expressed on eosinophils and induces apoptosis of eosinophils. In someembodiments, an antibody described herein binds to a human Siglec-8expressed on mast cells and depletes mast cells. In some embodiments, anantibody described herein binds to a human Siglec-8 expressed on mastcells and inhibits mast cell-mediated activity. In some embodiments, anantibody described herein binds to a human Siglec-8 expressed on mastcells and kills mast cells by ADCC activity. In some embodiments, anantibody described herein depletes mast cells and inhibits mast cellactivation. In some embodiments, an antibody herein depletes activatedeosinophils and inhibits mast cell activation.

Provided herein is an isolated anti-Siglec-8 antibody that binds tohuman Siglec-8 and non-human primate Siglec-8. Identification ofantibodies with primate cross-reactivity would be useful for preclinicaltesting of anti-Siglec-8 antibodies in non-human primates. In oneaspect, the invention provides antibodies that bind to a non-humanprimate Siglec-8. In one aspect, the invention provides antibodies thatbind to a human Siglec-8 and a non-human primate Siglec-8. In someembodiments, the non-human primate Siglec-8 comprises an amino acidsequence of SEQ ID NO:118 or a portion thereof In some embodiments, thenon-human primate Siglec-8 comprises an amino acid sequence of SEQ IDNO:119 or a portion thereof. In some embodiments, the non-human primateis a baboon (e.g., Papio anubis). In some embodiments, the antibody thatbinds to a human Siglec-8 and a non-human primate Siglec-8, binds to anepitope in Domain 1 of human Siglec-8. In a further embodiment, Domain 1of human Siglec-8 comprises the amino acid sequence of SEQ ID NO:112. Insome embodiments, the antibody that binds to a human Siglec-8 and anon-human primate Siglec-8, binds to an epitope in Domain 3 of humanSiglec-8. In a further embodiment, Domain 3 of human Siglec-8 comprisesthe amino acid sequence of SEQ ID NO:114. In some embodiments, theantibody that binds to a human Siglec-8 and a non-human primate Siglec-8is a humanized antibody, a chimeric antibody, or a human antibody. Insome embodiments, the antibody that binds to a human Siglec-8 and anon-human primate Siglec-8 is a murine antibody. In some embodiments,the antibody that binds to a human Siglec-8 and a non-human primateSiglec-8 is a human IgG1 antibody.

In one aspect, an anti-Siglec-8 antibody described herein is amonoclonal antibody. In one aspect, an anti-Siglec-8 antibody describedherein is an antibody fragment (including antigen-binding fragment),e.g., a Fab, Fab′-SH, Fv, scFv, or (Fab′)₂ fragment. In one aspect, ananti-Siglec-8 antibody described herein is a chimeric, humanized, orhuman antibody. In one aspect, any of the anti-Siglec-8 antibodiesdescribed herein are purified.

In one aspect, anti-Siglec-8 antibodies that compete with murine 2E2antibody and murine 2C4 antibody binding to Siglec-8 are provided.Anti-Siglec-8 antibodies that bind to the same epitope as murine 2E2antibody and murine 2C4 antibody are also provided. In one aspect,anti-Siglec-8 antibodies that compete with any anti-Siglec-8 antibodydescribed herein (e.g., HEKA, HEKF, 1C3, 1H10, 4F11) for binding toSiglec-8 are provided. Anti-Siglec-8 antibodies that bind to the sameepitope as any anti-Siglec-8 antibody described herein (e.g., HEKA,HEKF, 1C3, 1H10, 4F11) are also provided.

In one aspect of the invention, polynucleotides encoding anti-Siglec-8antibodies are provided. In certain embodiments, vectors comprisingpolynucleotides encoding anti-Siglec-8 antibodies are provided. Incertain embodiments, host cells comprising such vectors are provided. Inanother aspect of the invention, compositions comprising anti-Siglec-8antibodies or polynucleotides encoding anti-Siglec-8 antibodies areprovided. In certain embodiments, a composition of the invention is apharmaceutical formulation for the treatment of an eosinophil-mediateddisorder and/or mast cell-mediated disorder, such as those enumeratedherein.

In one aspect, provided herein is an anti-Siglec-8 antibody comprising1, 2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 2C4. Inone aspect, provided herein is an anti-Siglec-8 antibody comprising 1,2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 2E2. Insome embodiments, the HVR is a Kabat CDR or a Chothia CDR.

In one aspect, provided herein is an anti-Siglec-8 antibody comprising1, 2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 1C3. Inone aspect, provided herein is an anti-Siglec-8 antibody comprising 1,2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 4F11. Inone aspect, provided herein is an anti-Siglec-8 antibody comprising 1,2, 3, 4, 5, or 6 of the HVR sequences of the murine antibody 1H10. Insome embodiments, the HVR is a Kabat CDR or a Chothia CDR.

In some embodiments, the antibody described herein binds to an epitopein Domain 1 of human Siglec-8, wherein Domain 1 comprises the amino acidsequence of SEQ ID NO:112. In some embodiments, the antibody describedherein binds to an epitope in Domain 2 of human Siglec-8, wherein Domain2 comprises the amino acid sequence of SEQ ID NO:113. In someembodiments, the antibody described herein binds to an epitope in Domain3 of human Siglec-8, wherein Domain 3 comprises the amino acid sequenceof SEQ ID NO:114.

In some embodiments, the antibody described herein binds to a fusionprotein comprising the amino acid of SEQ ID NO:116 but not to a fusionprotein comprising the amino acid of SEQ ID NO:115. In some embodiments,the antibody described herein binds to a fusion protein comprising theamino acid of SEQ ID NO:117 but not to a fusion protein comprising theamino acid of SEQ ID NO:115. In some embodiments, the antibody describedherein binds to a fusion protein comprising the amino acid of SEQ IDNO:117 but not to a fusion protein comprising the amino acid of SEQ IDNO:116.

In one aspect, provided herein is an anti-Siglec-8 antibody comprising aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the aminoacid sequence of SEQ ID NO:63; and/or wherein the light chain variableregion comprises (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:66.

In one aspect, provided herein is an anti-Siglec-8 antibody comprising aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the aminoacid sequence selected from SEQ ID NOs:67-70; and/or wherein the lightchain variable region comprises (i) HVR-L1 comprising the amino acidsequence of SEQ ID NO:64, (ii) HVR-L2 comprising the amino acid sequenceof SEQ ID NO:65, and (iii) HVR-L3 comprising the amino acid sequence ofSEQ ID NO:66.

In one aspect, provided herein is an anti-Siglec-8 antibody comprising aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises (i) HVR-H1 comprising theamino acid sequence of SEQ ID NO:61, (ii) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:62, and (iii) HVR-H3 comprising the aminoacid sequence of SEQ ID NO:63; and/or wherein the light chain variableregion comprises (i) HVR-L1 comprising the amino acid sequence of SEQ IDNO:64, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:65,and (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:71.

In another aspect, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:61, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:62, and (iii) HVR-H3comprising the amino acid sequence selected from SEQ ID NOs:67-70;and/or wherein the light chain variable region comprises (i) HVR-L1comprising the amino acid sequence of SEQ ID NO:64, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO:65, and (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:71.

In another aspect, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:88, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:91, and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO:94; and/or a light chainvariable region comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:97, (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:100, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:103. In some embodiments, the antibody described herein binds to anepitope in Domain 2 of human Siglec-8, wherein Domain 2 comprises theamino acid sequence of SEQ ID NO: 113.

In another aspect, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:89, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:92, and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO:95; and/or a light chainvariable region comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:98, (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:101, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:104. In some embodiments, the antibody described herein binds to anepitope in Domain 3 of human Siglec-8, wherein Domain 3 comprises theamino acid sequence of SEQ ID NO: 114. In some embodiments, the antibodydescribed herein binds to human Siglec-8 and non-human primate Siglec-8.

In another aspect, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region comprises (i) HVR-H1comprising the amino acid sequence of SEQ ID NO:90, (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO:93, and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO:96; and/or a light chainvariable region comprising (i) HVR-L1 comprising the amino acid sequenceof SEQ ID NO:99, (ii) HVR-L2 comprising the amino acid sequence of SEQID NO:102, and (iii) HVR-L3 comprising the amino acid sequence of SEQ IDNO:105. In some embodiments, the antibody described herein binds to anepitope in Domain 1 of human Siglec-8, wherein Domain 1 comprises theamino acid sequence of SEQ ID NO: 112. In some embodiments, the antibodydescribed herein binds to human Siglec-8 and non-human primate Siglec-8.

An anti-Siglec-8 antibody described herein may comprise any suitableframework variable domain sequence, provided that the antibody retainsthe ability to bind human Siglec-8. As used herein, heavy chainframework regions are designated “HC-FR1-FR4,” and light chain frameworkregions are designated “LC-FR1-FR4.” In some embodiments, theanti-Siglec-8 antibody comprises a heavy chain variable domain frameworksequence of SEQ ID NO:26, 34, 38, and 45 (HC-FR1, HC-FR2, HC-FR3, andHC-FR4, respectively). In some embodiments, the anti-Siglec-8 antibodycomprises a light chain variable domain framework sequence of SEQ IDNO:48, 51, 55, and 60 (LC-FR1, LC-FR2, LC-FR3, and LC-FR4,respectively). In some embodiments, the anti-Siglec-8 antibody comprisesa light chain variable domain framework sequence of SEQ ID NO:48, 51,58, and 60 (LC-FR1, LC-FR2, LC-FR3, and LC-FR4, respectively).

In one embodiment, an anti-Siglec-8 antibody comprises a heavy chainvariable domain comprising a framework sequence and hypervariableregions, wherein the framework sequence comprises the HC-FR1-HC-FR4sequences SEQ ID NOs:26-29 (HC-FR1), SEQ ID NOs:31-36 (HC-FR2), SEQ IDNOs:38-43 (HC-FR3), and SEQ ID NOs:45 or 46 (HC-FR4), respectively; theHVR-H1 comprises the amino acid sequence of SEQ ID NO:61; the HVR-H2comprises the amino acid sequence of SEQ ID NO:62; and the HVR-H3comprises an amino acid sequence of SEQ ID NO:63. In one embodiment, ananti-Siglec-8 antibody comprises a heavy chain variable domaincomprising a framework sequence and hypervariable regions, wherein theframework sequence comprises the HC-FR1-HC-FR4 sequences SEQ IDNOs:26-29 (HC-FR1), SEQ ID NOs:31-36 (HC-FR2), SEQ ID NOs:38-43(HC-FR3), and SEQ ID NOs:45 or 46 (HC-FR4), respectively; the HVR-H1comprises the amino acid sequence of SEQ ID NO:61; the HVR-H2 comprisesthe amino acid sequence of SEQ ID NO:62; and the HVR-H3 comprises anamino acid sequence selected from SEQ ID NOs:67-70. In one embodiment,an anti-Siglec-8 antibody comprises a light chain variable domaincomprising a framework sequence and hypervariable regions, wherein theframework sequence comprises the LC-FR1-LC-FR4 sequences SEQ ID NOs:48or 49 (LC-FR1), SEQ ID NOs:51-53 (LC-FR2), SEQ ID NOs:55-58 (LC-FR3),and SEQ ID NO:60 (LC-FR4), respectively; the HVR-L1 comprises the aminoacid sequence of SEQ ID NO:64; the HVR-L2 comprises the amino acidsequence of SEQ ID NO:65; and the HVR-L3 comprises an amino acidsequence of SEQ ID NO:66. In one embodiment, an anti-Siglec-8 antibodycomprises a light chain variable domain comprising a framework sequenceand hypervariable regions, wherein the framework sequence comprises theLC-FR1-LC-FR4 sequences SEQ ID NOs:48 or 49 (LC-FR1), SEQ ID NOs:51-53(LC-FR2), SEQ ID NOs:55-58 (LC-FR3), and SEQ ID NO:60 (LC-FR4),respectively; the HVR-L1 comprises the amino acid sequence of SEQ IDNO:64; the HVR-L2 comprises the amino acid sequence of SEQ ID NO:65; andthe HVR-L3 comprises an amino acid sequence of SEQ ID NO:71. In oneembodiment of these antibodies, the heavy chain variable domaincomprises an amino acid sequence selected from SEQ ID NOs:2-10 and thelight chain variable domain comprises and amino acid sequence selectedfrom SEQ ID NOs:16-22. In one embodiment of these antibodies, the heavychain variable domain comprises an amino acid sequence selected from SEQID NOs:2-10 and the light chain variable domain comprises and amino acidsequence selected from SEQ ID NOs:23 or 24. In one embodiment of theseantibodies, the heavy chain variable domain comprises an amino acidsequence selected from SEQ ID NOs:11-14 and the light chain variabledomain comprises and amino acid sequence selected from SEQ ID NOs:16-22.In one embodiment of these antibodies, the heavy chain variable domaincomprises an amino acid sequence selected from SEQ ID NOs:11-14 and thelight chain variable domain comprises and amino acid sequence selectedfrom SEQ ID NOs:23 or 24. In one embodiment of these antibodies, theheavy chain variable domain comprises an amino acid sequence of SEQ IDNO:6 and the light chain variable domain comprises and amino acidsequence of SEQ ID NO:16. In one embodiment of these antibodies, theheavy chain variable domain comprises an amino acid sequence of SEQ IDNO:6 and the light chain variable domain comprises and amino acidsequence of SEQ ID NO:21.

In some embodiments, the heavy chain HVR sequences comprise thefollowing:

  a) HVR-H1 (SEQ ID NO: 61) (IYGAH); b) HVR-H2 (SEQ ID NO: 62)(VIWAGGSTNYNSALMS); and c) HVR-H3 (SEQ ID NO: 63) (DGSSPYYYSMEY;(SEQ ID NO: 67) DGSSPYYYGMEY; (SEQ ID NO: 68) DGSSPYYYSMDY;(SEQ ID NO: 69) DGSSPYYYSMEV; or (SEQ ID NO: 70) GSSPYYYGMDV).

In some embodiments, the heavy chain HVR sequences comprise thefollowing:

  a) HVR-H1 (SEQ ID NO: 88) (SYAMS; (SEQ ID NO: 89) DYYMY; or(SEQ ID NO: 90) SSWMN); b) HVR-H2 (SEQ ID NO: 91) (IISSGGSYTYYSDSVKG;(SEQ ID NO: 92) RIAPEDGDTEYAPKFQG; or (SEQ ID NO: 93)QIYPGDDYTNYNGKFKG); and c) HVR-H3 (SEQ ID NO: 94) (HETAQAAWFAY;(SEQ ID NO: 95) EGNYYGSSILDY; or (SEQ ID NO: 96) LGPYGPFAD).

In some embodiments, the heavy chain FR sequences comprise thefollowing:

  a) HC-FR1 (SEQ ID NO: 26) (EVQLVESGGGLVQPGGSLRLSCAASGFSLT;(SEQ ID NO: 27) EVQLVESGGGLVQPGGSLRLSCAVSGFSLT; (SEQ ID NO: 28)QVQLQESGPGLVKPSETLSLTCTVSGGSIS; or (SEQ ID NO: 29)QVQLQESGPGLVKPSETLSLTCTVSGFSLT); b) HC-FR2 (SEQ ID NO: 31)(WVRQAPGKGLEWVS; (SEQ ID NO: 32) WVRQAPGKGLEWLG; (SEQ ID NO: 33)WVRQAPGKGLEWLS; (SEQ ID NO: 34) WVRQAPGKGLEWVG; (SEQ ID NO: 35)WIRQPPGKGLEWIG; or (SEQ ID NO: 36) WVRQPPGKGLEWLG); c) HC-FR3(SEQ ID NO: 38) (RFTISKDNSKNTVYLQMNSLRAEDTAVYYCAR; (SEQ ID NO: 39)RLSISKDNSKNTVYLQMNSLRAEDTAVYYCAR; (SEQ ID NO: 40)RLTISKDNSKNTVYLQMNSLRAEDTAVYYCAR; (SEQ ID NO: 41)RFSISKDNSKNTVYLQMNSLRAEDTAVYYCAR; (SEQ ID NO: 42)RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR; or (SEQ ID NO: 43)RLSISKDNSKNQVSLKLSSVTAADTAVYYCAR); and d) HC-FR4 (SEQ ID NO: 45)(WGQGTTVTVSS; or (SEQ ID NO: 46) WGQGTLVTVSS).

In some embodiments, the light chain HVR sequences comprise thefollowing:

  a) HVR-L1 (SEQ ID NO: 64) (SATSSVSYMH); b) HVR-L2 (SEQ ID NO: 65)(STSNLAS); and c) HVR-L3 (SEQ ID NO: 66) (QQRSSYPFT; or (SEQ ID NO: 71)QQRSSYPYT)

In some embodiments, the light chain HVR sequences comprise thefollowing:

  a) HVR-L1 (SEQ ID NO: 97) (SASSSVSYMH; (SEQ ID NO: 98) RASQDITNYLN; or(SEQ ID NO: 99) SASSSVSYMY); b) HVR-L2 (SEQ ID NO: 100) (DTSKLAY;(SEQ ID NO: 101) FTSRLHS; or (SEQ ID NO: 102) DTSSLAS); and c) HVR-L3(SEQ ID NO: 103) (QQWSSNPPT; (SEQ ID NO: 104) QQGNTLPWT; or(SEQ ID NO: 105) QQWNSDPYT).

In some embodiments, the light chain FR sequences comprise thefollowing:

  a) LC-FR1 (SEQ ID NO: 48) (EIVLTQSPATLSLSPGERATLSC; or (SEQ ID NO: 49)EIILTQSPATLSLSPGERATLSC); b) LC-FR2 (SEQ ID NO: 51) (WFQQKPGQAPRLLIY;(SEQ ID NO: 52) WFQQKPGQAPRLWIY; or (SEQ ID NO: 53) WYQQKPGQAPRLLIY);c) LC-FR3 (SEQ ID NO: 55) (GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC;(SEQ ID NO: 56) GVPARFSGSGSGTDYTLTISSLEPEDFAVYYC; (SEQ ID NO: 57)GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC; or (SEQ ID NO: 58)GIPARFSGSGSGTDYTLTISSLEPEDFAVYYC); and d) LC-FR4 (SEQ ID NO: 60)(FGPGTKLDIK).

In some embodiments, provided herein is an anti-Siglec-8 antibody (e.g.,a humanized anti-Siglec-8) antibody that specifically binds to humanSiglec-8, wherein the antibody comprises a heavy chain variable regionand a light chain variable region, wherein the antibody comprises:

(a) heavy chain variable domain comprising:

(1) an HC-FR1 comprising the amino add sequence selected from SEQ IDNOs:26-29;

(2) an HVR-H1 comprising the amino acid sequence of SEQ ID NO:61;

(3) an HC-FR2 comprising the amino acid sequence selected from SEQ IDNOs:31-36;

(4) an HVR-H2 comprising the amino acid sequence of SEQ ID NO:62;

(5) an HC-FR3 comprising the amino acid sequence selected from SEQ IDNOs:38-43;

(6) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; and

(7) an HC-FR4 comprising the amino acid sequence selected from SEQ 1DNOs:45-46,

and/or

(b) a light chain variable domain comprising:

(1) an LC-FRI comprising the amino acid sequence selected from SEQ IDNOs:48-49;

(2) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:64;

(3) an LC-FR2 comprising the amino acid sequence selected from SEQ IDNOs:51-53;

(4) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:65;

(5) an LC-FR3 comprising the amino acid sequence selected from SEQ IDNOs:55-58;

(6) an HVR-L3 comprising the amino acid sequence of SEQ ID NO:66; and

(7) an LC-FR4 comprising the amino acid sequence of SEQ ID NO:60.

In one aspect, provided herein is an anti-Siglec-8 antibody comprising aheavy chain variable domain selected from SEQ ID NOs:2-10 and/orcomprising a light chain variable domain selected from SEQ ID NOs:16-22.In one aspect, provided herein is an anti-Siglec-8 antibody comprising aheavy chain variable domain selected from SEQ ID NOs:2-10 and/orcomprising a light chain variable domain selected from SEQ ID NO:23 or24. In one aspect, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable domain selected from SEQ ID NOs:11-14and/or comprising a light chain variable domain selected from SEQ IDNOs:16-22. In one aspect, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable domain selected from SEQ ID NOs:11-14and/or comprising a light chain variable domain selected from SEQ IDNO:23 or 24. In one aspect, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable domain of SEQ ID NO:6 and/orcomprising a light chain variable domain selected from SEQ ID NO:16 or21.

In one aspect, provided herein is an anti-Siglec-8 antibody comprising aheavy chain variable domain selected from SEQ ID NOs:106-108 and/orcomprising a light chain variable domain selected from SEQ IDNOs:109-111. In one aspect, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable domain of SEQ ID NO:106 and/orcomprising a light chain variable domain of SEQ ID NO:109. In oneaspect, provided herein is an anti-Siglec-8 antibody comprising a heavychain variable domain of SEQ ID NO:107 and/or comprising a light chainvariable domain of SEQ ID NO:110. In one aspect, provided herein is ananti-Siglec-8 antibody comprising a heavy chain variable domain of SEQID NO:108 and/or comprising a light chain variable domain of SEQ IDNO:111.

In some embodiments, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to an amino acid sequence selected from SEQ IDNOs:2-14. In some embodiments, provided herein is an anti-Siglec-8antibody comprising a heavy chain variable domain comprising an aminoacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to an amino acid sequence selected fromSEQ ID NOs:106-108. In some embodiments, an amino acid sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity contains substitutions, insertions, or deletions relative tothe reference sequence, but an antibody comprising that amino acidsequence retains the ability to bind to human Siglec-8. In someembodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3,4, or 5 amino acids) occur in regions outside the HVRs (i.e., in theFRs). In some embodiments, an anti-Siglec-8 antibody comprises a heavychain variable domain comprising an amino acid sequence of SEQ ID NO:6.In some embodiments, an anti-Siglec-8 antibody comprises a heavy chainvariable domain comprising an amino acid sequence selected from SEQ IDNOs:106-108.

In some embodiments, provided herein is an anti-Siglec-8 antibodycomprising a light chain variable domain comprising an amino acidsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity to an amino acid sequence selected from SEQ IDNOs:16-24. In some embodiments, provided herein is an anti-Siglec-8antibody comprising a light chain variable domain comprising an aminoacid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to an amino acid sequence selected fromSEQ ID NOs:109-111. In some embodiments, an amino acid sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity contains substitutions, insertions, or deletions relative tothe reference sequence, but an antibody comprising that amino acidsequence retains the ability to bind to human Siglec-8. In someembodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3,4, or 5 amino acids) occur in regions outside the HVRs (i.e., in theFRs). In some embodiments, an anti-Siglec-8 antibody comprises a lightchain variable domain comprising an amino acid sequence of SEQ ID NO:16or 21. In some embodiments, an anti-Siglec-8 antibody comprises a heavychain variable domain comprising an amino acid sequence selected fromSEQ ID NOs:109-111.

In some embodiments, provided herein is an anti-Siglec-8 antibodycomprises a heavy chain variable domain as depicted in FIG. 1 or FIG. 3.

In some embodiments, provided herein is an anti-Siglec-8 antibodycomprises a light chain variable domain as depicted in FIG. 2 or FIG. 3.

In one aspect, the invention provides an anti-Siglec-8 antibodycomprising (a) one, two, or three VH HVRs selected from those shown inFIG. 1 or FIG. 3 and/or (b) one, two, or three VL HVRs selected fromthose shown in FIG. 2 or FIG. 3. In one aspect, the invention providesan anti-Siglec-8 antibody comprising a heavy chain variable domainselected from those shown in FIG. 1 or FIG. 3 and a light chain variabledomain selected from those shown in FIG. 2 or FIG. 3.

In some embodiments, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain variable domain and/or a light chain variabledomain of an antibody shown in Table 3, for example, HAKA antibody, HAKBantibody, HAKC antibody, etc.

There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM,having heavy chains designated α, δ, ε, γ and μ, respectively. The γ andα classes are further divided into subclasses e.g., humans express thefollowing subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. IgG1antibodies can exist in multiple polymorphic variants termed allotypes(reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any ofwhich are suitable for use in some of the embodiments herein. Commonallotypic variants in human populations are those designated by theletters a,f,n,z or combinations thereof. In any of the embodimentsherein, the antibody may comprise a heavy chain Fc region comprising ahuman IgG Fc region. In further embodiments, the human IgG Fc regioncomprises a human IgG1 or IgG4. In some embodiments, the human IgG4comprises the amino acid substitution S228P, wherein the amino acidresidues are numbered according to the EU index as in Kabat. In someembodiments, the human IgG1 comprises the amino acid sequence of SEQ IDNO:78. In some embodiments, the human IgG4 comprises the amino acidsequence of SEQ ID NO:79.

In some embodiments, provided herein is an anti-Siglec-8 antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO:75; and/or a light chain comprising the amino acid sequence selectedfrom SEQ ID NOs:76 or 77. In some embodiments, the antibody may comprisea heavy chain comprising the amino acid sequence of SEQ ID NO:87; and/ora light chain comprising the amino acid sequence of SEQ ID NO:76. Insome embodiments, the anti-Siglec-8 antibody depletes mast cells andinhibits mast cell activation. In some embodiments, the anti-Siglec-8antibody depletes activated eosinophils and inhibits mast cellactivation.

1. Antibody Affinity

In some aspects, an anti-Siglec-8 antibody described herein binds tohuman Siglec-8 with about the same or higher affinity and/or higheravidity as compared mouse antibody 2E2 and/or mouse antibody 2C4. Incertain embodiments, an anti-Siglec-8 antibody provided herein has adissociation constant (Kd) of ≤1 μM, ≤150 nM, ≤100 nM, ≤50 nM, ≤10 nM,≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In some embodiments,an anti-Siglec-8 antibody described herein binds to human Siglec-8 atabout 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold,about 6-fold, about 7-fold, about 8-fold, about 9-fold or about 10-foldhigher affinity than mouse antibody 2E2 and/or mouse antibody 2C4. Insome embodiments herein, the anti-Siglec-8 antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:6;and/or a light chain variable region comprising the amino acid sequenceselected from SEQ ID NOs:16 or 21.

In one embodiment, the binding affinity of the anti-Siglec-8 antibodycan be determined by a surface plasmon resonance assay. For example, theKd or Kd value can be measured by using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. withimmobilized antigen CM5 chips at ˜10 response units (RU). Briefly,carboxymethylated dextran biosensor chips (CM5, BIAcore® Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Capture antibodies (e.g., anti-human-Fc) arediluted with 10 mM sodium acetate, pH 4.8, before injection at a flowrate of 30 μl/minute and further immobilized with an anti-Siglec-8antibody. For kinetics measurements, two-fold serial dilutions ofdimeric Siglec-8 are injected in PBS with 0.05% Tween 20 (PBST) at 25°C. at a flow rate of approximately 25 μl/min. Association rates (kon)and dissociation rates (koff) are calculated using a simple one-to-oneLangmuir binding model (BIAcore® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiokoff/kon. See, e.g., Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881.

In another embodiment, biolayer interferometry may be used to determinethe affinity of anti-Siglec-8 antibodies against Siglec-8. In anexemplary assay, Siglec-8-Fc tagged protein is immobilized ontoanti-human capture sensors, and incubated with increasing concentrationsof mouse, chimeric, or humanized anti-Siglec-8 Fab fragments to obtainaffinity measurements using an instrument such as, for example, theOctet Red 384 System (ForteBio).

The binding affinity of the anti-Siglec-8 antibody can, for example,also be determined by the Scatchard analysis described in Munson et al.,Anal. Biochem., 107:220 (1980) using standard techniques well known inthe relevant art. See also Scatchard, G., Ann. N.Y. Acad. Sci. 51:660(1947).

2. Antibody Avidity

In one embodiment, the binding avidity of the anti-Siglec-8 antibody canbe determined by a surface plasmon resonance assay. For example, the Kdor Kd value can be measured by using a BIAcore T100. Capture antibodies(e.g., goat-anti-human-Fc and goat-anti-mouse-Fc) are immobilized on aCM5 chip. Flow-cells can be immobilized with anti-human or withanti-mouse antibodies. The assay is conducted at a certain temperatureand flow rate, for example, at 25° C. at a flow rate of 30 μl/min.Dimeric Siglec-8 is diluted in assay buffer at various concentrations,for example, at a concentration ranging from 15 nM to 1.88 pM.Antibodies are captured and high performance injections are conducted,followed by dissociations. Flow cells are regenerated with a buffer, forexample, 50 mM glycine pH 1.5. Results are blanked with an emptyreference cell and multiple assay buffer injections, and analyzed with1:1 global fit parameters.

3. Competition Assays

Competition assays can be used to determine whether two antibodies bindthe same epitope by recognizing identical or sterically overlappingepitopes or one antibody competitively inhibits binding of anotherantibody to the antigen. These assays are known in the art. Typically,antigen or antigen expressing cells is immobilized on a multi-well plateand the ability of unlabeled antibodies to block the binding of labeledantibodies is measured. Common labels for such competition assays areradioactive labels or enzyme labels. In some embodiments, ananti-Siglec-8 antibody described herein competes with a 2E2 antibodydescribed herein, for binding to the epitope present on the cell surfaceof a cell (e.g., an eosinophil). In some embodiments, an anti-Siglec-8antibody described herein competes with an antibody comprising a heavychain variable domain comprising the amino acid sequence of SEQ ID NO:1,and a light chain variable region comprising the amino acid sequence ofSEQ ID NO:15, for binding to the epitope present on the cell surface ofa cell (e.g., an eosinophil). In some embodiments, an anti-Siglec-8antibody described herein competes with a 2C4 antibody described herein,for binding to the epitope present on the cell surface of a cell (e.g.,an eosinophil). In some embodiments, an anti-Siglec-8 antibody describedherein competes with an antibody comprising a heavy chain variabledomain comprising the amino acid sequence of SEQ ID NO:2 (as found inU.S. Pat. No. 8,207,305), and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:4 (as found in U.S. Pat. No.8,207,305), for binding to the epitope present on the cell surface of acell (e.g., an eosinophil).

4. Thermal Stability

In some aspects, an anti-Siglec-8 described herein has a meltingtemperature (Tm) of at least about 70° C., at least about 71° C., or atleast about 72° C. in a thermal shift assay. In an exemplary thermalshift assay, samples comprising a humanized anti-Siglec-8 antibody areincubated with a fluorescent dye (Sypro Orange) for 71 cycles with 1° C.increase per cycle in a qPCR thermal cycler to determine the Tm. In someembodiments herein, the anti-Siglec-8 antibody has a similar or higherTm as compared to mouse 2E2 antibody and/or mouse 2C4 antibody. In someembodiments herein, the anti-Siglec-8 antibody comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:6;and/or a light chain variable region comprising the amino acid sequenceselected from SEQ ID NOs:16 or 21. In some embodiments, theanti-Siglec-8 antibody has the same or higher Tm as compared to achimeric 2C4 antibody. In some embodiments, the anti-Siglec-8 antibodyhas the same or higher Tm as compared to an antibody having a heavychain comprising the amino acid sequence of SEQ ID NO:84 and a lightchain comprising the amino acid sequence of SEQ ID NO:85.

5. Biological Activity Assays

In some aspects, an anti-Siglec-8 described herein induces apoptosis ofeosinophils. In some other aspects, an anti-Siglec-8 described hereindepletes mast cells. Assays for assessing apoptosis of cells are wellknown in the art, for example staining with Annexin V and the TUNNELassay. In an exemplary cell apoptosis assay, fresh buffy coat from ablood sample is resuspended in media and plated in a 96-well U-bottomplate. A series of serial 5-fold dilutions of anti-Siglec-8 antibody isadded to each well and the plate is incubated at 37° C. at 5% CO₂ forgreater than four hours. The cells are fixed with paraformaldehydediluted in PBS and stained with conjugated antibodies specific foreosinophils for detection using a microscope. The eosinophil populationin the total peripheral blood leukocytes is evaluated when the buffycoat is incubated in the presence of the anti-Siglec-8 antibody ascompared to when the buffy coat is not incubated in the presence of theanti-Siglec-8 antibody. In another exemplary assay, eosinophils purifiedfrom a blood sample (e.g., Miltenyi Eosinophil Isolation Kit) areresuspended in media and cultured in the presence or absence of IL-5overnight. The cultured eosinophils are subsequently harvested bycentrifugation, resuspended in media, and plated in a 96-well U-bottomplate. A series of serial 5-fold dilutions of anti-Siglec-8 antibody isadded to each well and the plate is incubated at 37° C. at 5% CO₂ forgreater than four hours. The cells are fixed and stained with Annexin-Vusing standard techniques well known in the art the number ofeosinophils is detected using a microscope. The eosinophil population inthe sample is evaluated when the purified cells are incubated in thepresence of the anti-Siglec-8 antibody as compared to when the purifiedcells are not incubated in the presence of the anti-Siglec-8 antibody.

In some aspects, an anti-Siglec-8 antibody described herein induces ADCCactivity. In some other aspects, an anti-Siglec-8 antibody describedherein kills mast cells expressing Siglec-8 by ADCC activity. In someembodiments, a composition comprises non-fucosylated (i.e.,afucosylated) anti-Siglec-8 antibodies. In some embodiments, acomposition comprising non-fucosylated anti-Siglec-8 antibodiesdescribed herein enhances ADCC activity as compared to a compositioncomprising partially fucosylated anti-Siglec-8 antibodies. Assays forassessing ADCC activity are well known in the art and described herein.In an exemplary assay, to measure ADCC activity, effector cells andtarget cells are used. Examples of effector cells include natural killer(NK) cells, large granular lymphocytes (LGL), lymphokine-activatedkiller (LAK) cells and PBMC comprising NK and LGL, or leukocytes havingFc receptors on the cell surfaces, such as neutrophils, eosinophils andmacrophages. The target cell is any cell which expresses on the cellsurface antigens that antibodies to be evaluated can recognize. Anexample of such a target cell is an eosinophil which expresses Siglec-8on the cell surface. Another example of such a target cell is a mastcell which expresses Siglec-8 on the cell surface. Target cells arelabeled with a reagent that enables detection of cytolysis. Examples ofreagents for labeling include a radio-active substance such as sodiumchromate (Na₂ ⁵¹CrO₄). See, e.g., Immunology, 14, 181 (1968); J.Immunol. Methods, 172, 227 (1994); and J. Immunol. Methods, 184, 29(1995).

In some aspects, an anti-Siglec-8 antibody described herein inhibitsmast cell-mediated activities. Mast cell tryptase has been used as abiomarker for total mast cell number and activation. For example, totaland active tryptase as well as histamine, N-methyl histamine, and11-beta-prostaglandin F2 can be measured in blood or urine to assess thereduction in mast cells. See, e.g., U.S. Patent Application PublicationNo. US 20110293631 for an exemplary mast cell activity assay. Assaysdescribed in Example 2 herein can also be used to assess ADCC andapoptotic activity of anti-Siglec-8 antibodies on mast cells.

6. Fusion Protein Binding Assays

Binding assays with fusion proteins can be used to determine the epitoperecognized by an antibody. Assays using fusion proteins for epitopemapping are known in the art. For example, a fusion protein comprising aportion of a Siglec-8 protein fused to a human Ig-Fc is immobilized on amulti-well plate and the ability of antibodies to bind to the fusionprotein is measured. In some embodiments, an anti-Siglec-8 antibodydescribed herein binds to a fusion protein comprising the amino acidsequence of SEQ ID NO:115. In some embodiments, an anti-Siglec-8antibody described herein binds to a fusion protein comprising the aminoacid sequence of SEQ ID NO:116. In some embodiments, an anti-Siglec-8antibody described herein binds to a fusion protein comprising the aminoacid sequence of SEQ ID NO:117. In some embodiments, an anti-Siglec-8antibody described herein binds to a fusion protein comprising the aminoacid sequence of SEQ ID NO:118.

III. Antibody Preparation

The antibody described herein is prepared using techniques available inthe art for generating antibodies, exemplary methods of which aredescribed in more detail in the following sections.

1. Antibody Fragments

The present invention encompasses antibody fragments. Antibody fragmentsmay be generated by traditional means, such as enzymatic digestion, orby recombinant techniques. In certain circumstances there are advantagesof using antibody fragments, rather than whole antibodies. For a reviewof certain antibody fragments, see Hudson et al. (2003) Nat. Med.9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂fragments(Carter et al., Bio/Technology 10: 163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

2. Humanized Antibodies

The invention encompasses humanized antibodies. Various methods forhumanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter (Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen etal. (1988) Science 239:1534-1536), by substituting hypervariable regionsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567) wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species. In practice, humanized antibodies are typicallyhuman antibodies in which some hypervariable region residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies can be important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent (e.g., mouse) antibody is screenedagainst the entire library of known human variable-domain sequences. Thehuman sequence which is closest to that of the rodent is then acceptedas the human framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further generally desirable that antibodies be humanized withretention of high affinity for the antigen and other favorablebiological properties. To achieve this goal, according to one method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those, skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

3. Human Antibodies

Human anti-Siglec-8 antibodies of the invention can be constructed bycombining Fv clone variable domain sequence(s) selected fromhuman-derived phage display libraries with known human constant domainsequences(s). Alternatively, human monoclonal anti-Siglec-8 antibodiesof the invention can be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human (e.g., rodent) antibodies, where the human antibody hassimilar affinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e., the epitope governs the choiceof the human chain partner. When the process is repeated in order toreplace the remaining non-human chain, a human antibody is obtained (seePCT WO 93/06213 published Apr. 1, 1993). Unlike traditional humanizationof non-human antibodies by CDR grafting, this technique providescompletely human antibodies, which have no FR or CDR residues ofnon-human origin.

4. Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different antigens. In certainembodiments, bispecific antibodies are human or humanized antibodies. Incertain embodiments, one of the binding specificities is for Siglec-8and the other is for any other antigen. In certain embodiments,bispecific antibodies may bind to two different epitopes of Siglec-8.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express Siglec-8. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art. SeeMilstein and Cuello, Nature, 305: 537 (1983),WO 93/08829 published May13, 1993, and Traunecker et al., EMBO J., 10: 3655 (1991). For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986). Bispecific antibodiesinclude cross-linked or “heteroconjugate” antibodies. For example, oneof the antibodies in the heteroconjugate can be coupled to avidin, theother to biotin. Heteroconjugate antibodies may be made using anyconvenient cross-linking method. Suitable cross-linking agents are wellknown in the art, and are disclosed in U.S. Pat. No. 4,676,980, alongwith a number of cross-linking techniques.

5. Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

6. Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (e.g.,alanine or polyalanine) to affect the interaction of the amino acidswith antigen. Those amino acid locations demonstrating functionalsensitivity to the substitutions then are refined by introducing furtheror other variants at, or for, the sites of substitution. Thus, while thesite for introducing an amino acid sequence variation is predetermined,the nature of the mutation per se need not be predetermined. Forexample, to analyze the performance of a mutation at a given site, alascanning or random mutagenesis is conducted at the target codon orregion and the expressed immunoglobulins are screened for the desiredactivity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme or a polypeptide which increasesthe serum half-life of the antibody.

In certain embodiments, an antibody of the invention is altered toincrease or decrease the extent to which the antibody is glycosylated.Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of a carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition or deletion of glycosylation sites to the antibody isconveniently accomplished by altering the amino acid sequence such thatone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites) is created or removed. The alteration may also bemade by the addition, deletion, or substitution of one or more serine orthreonine residues to the sequence of the original antibody (forO-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

In certain embodiments, a glycosylation variant comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucoseor has reduced fucose. Such variants have improved ADCC function.Optionally, the Fc region further comprises one or more amino acidsubstitutions therein which further improve ADCC, for example,substitutions at positions 298, 333, and/or 334 of the Fc region (Eunumbering of residues). Examples of publications related to“defucosylated” or “fucose-deficient” antibodies include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)), and cellsoverexpressing β1,4-N-acetylglycosminyltransferase III (GnT-III) andGolgi μ-mannosidase II (ManII).

Antibodies are contemplated herein that have reduced fucose relative tothe amount of fucose on the same antibody produced in a wild-type CHOcell. For example, the antibody has a lower amount of fucose than itwould otherwise have if produced by native CHO cells (e.g., a CHO cellthat produce a native glycosylation pattern, such as, a CHO cellcontaining a native FUT8 gene). In certain embodiments, an anti-Siglec-8antibody provided herein is one wherein less than about 50%, 40%, 30%,20%, 10%, 5% or 1% of the N-linked glycans thereon comprise fucose. Incertain embodiments, an anti-Siglec-8 antibody provided herein is onewherein none of the N-linked glycans thereon comprise fucose, i.e.,wherein the antibody is completely without fucose, or has no fucose oris non-fucosylated or is afucosylated. The amount of fucose can bedetermined by calculating the average amount of fucose within the sugarchain at Asn297, relative to the sum of all glycostructures attached toAsn297 (e.g., complex, hybrid and high mannose structures) as measuredby MALDI-TOF mass spectrometry, as described in WO 2008/077546, forexample. Asn297 refers to the asparagine residue located at aboutposition 297 in the Fc region (Eu numbering of Fc region residues);however, Asn297 may also be located about ±3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. In some embodiments, at leastone or two of the heavy chains of the antibody is non-fucosylated.

In one embodiment, the antibody is altered to improve its serumhalf-life. To increase the serum half-life of the antibody, one mayincorporate a salvage receptor binding epitope into the antibody(especially an antibody fragment) as described in U.S. Pat. No.5,739,277, for example. As used herein, the term “salvage receptorbinding epitope” refers to an epitope of the Fc region of an IgGmolecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible forincreasing the in vivo serum half-life of the IgG molecule (US2003/0190311, U.S. Pat. Nos. 6,821,505; 6,165,745; 5,624,821; 5,648,260;6,165,745; 5,834,597).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. Sites of interest for substitutionalmutagenesis include the hypervariable regions, but FR alterations arealso contemplated. Conservative substitutions are shown in Table 1 underthe heading of “preferred substitutions.” If such substitutions resultin a desirable change in biological activity, then more substantialchanges, denominated “exemplary substitutions” in Table 1, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE 1 Preferred Original Residue Exemplary Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Leu Norleucine Leu (L) Norleucine;Ile; Val; Met; Ile Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leu Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or c) the bulk of the side chain. Amino acids may begrouped according to similarities in the properties of their side chains(in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, WorthPublishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Len (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (Q)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), His (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, into theremaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g., a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have modified (e.g., improved) biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g., 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibodies thusgenerated are displayed from filamentous phage particles as fusions toat least part of a phage coat protein (e.g., the gene III product ofM13) packaged within each particle. The phage-displayed variants arethen screened for their biological activity (e.g., binding affinity). Inorder to identify candidate hypervariable region sites for modification,scanning mutagenesis (e.g., alanine scanning) can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and antigen. Such contact residuesand neighboring residues are candidates for substitution according totechniques known in the art, including those elaborated herein. Oncesuch variants are generated, the panel of variants is subjected toscreening using techniques known in the art, including those describedherein, and antibodies with superior properties in one or more relevantassays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of antibodies of the invention, thereby generating an Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine. In some embodiments,the Fc region variant comprises a human IgG4 Fc region. In a furtherembodiment, the human IgG4 Fc region comprises the amino acidsubstitution S228P, wherein the amino acid residues are numberedaccording to the EU index as in Kabat.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody of the invention maycomprise one or more alterations as compared to the wild typecounterpart antibody, e.g. in the Fc region. These antibodies wouldnonetheless retain substantially the same characteristics required fortherapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO94/29351concerning other examples of Fc region variants. WO00/42072 (Presta) andWO 2004/056312 (Lowman) describe antibody variants with improved ordiminished binding to FcRs. The content of these patent publications arespecifically incorporated herein by reference. See, also, Shields et al.J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodies with increasedhalf-lives and improved binding to the neonatal Fc receptor (FcRn),which is responsible for the transfer of maternal IgGs to the fetus(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). Theseantibodies comprise an Fc region with one or more substitutions thereinwhich improve binding of the Fc region to FcRn. Polypeptide variantswith altered Fc region amino acid sequences and increased or decreasedC1q binding capability are described in U.S. Pat. No. 6,194,551B1,WO99/51642. The contents of those patent publications are specificallyincorporated herein by reference. See, also, Idusogie et al. J. Immunol.164: 4178-4184 (2000).

7. Vectors, Host Cells, and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, hostcells are of either prokaryotic or eukaryotic (generally mammalian)origin. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species.

Generating Antibodies Using Prokaryotic Host Cells:

a) Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes-encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. colitrxB-strains) provide cytoplasm conditions that are favorable fordisulfide bond formation, thereby permitting proper folding and assemblyof expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Antibodies of the invention can also be produced by using an expressionsystem in which the quantitative ratio of expressed polypeptidecomponents can be modulated in order to maximize the yield of secretedand properly assembled antibodies of the invention. Such modulation isaccomplished at least in part by simultaneously modulating translationalstrengths for the polypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence. In certain embodiments, changes in the nucleotidesequence are silent. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

In one embodiment, a set of vectors is generated with a range of TIRstrengths for each cistron therein. This limited set provides acomparison of expression levels of each chain as well as the yield ofthe desired antibody products under various TIR strength combinations.TIR strengths can be determined by quantifying the expression level of areporter gene as described in detail in Simmons et al. U.S. Pat. No.5,840,523. Based on the translational strength comparison, the desiredindividual TIRs are selected to be combined in the expression vectorconstructs of the invention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli ), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kanR (U.S. Pat. No. 5,639,635). Other strains and derivatives thereof,such as E. coli 294 (ATCC 31,446), E. coli B, E. coliλ 1776 (ATCC31,537) and E. coli RV308(ATCC 31,608) are also suitable. These examplesare illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

b) Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. Incertain embodiments, for E. coli growth, growth temperatures range fromabout 20° C. to about 39° C.; from about 25° C. to about 37° C.; orabout 30° C. The pH of the medium may be any pH ranging from about 5 toabout 9, depending mainly on the host organism. In certain embodiments,for E. coli , the pH is from about 6.8 to about 7.4, or about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. In certain embodiments, thephosphate-limiting medium is the C.R.A.P. medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, and in certain embodiments, about 1,000 to 100,000 liters ofcapacity. These fermentors use agitator impellers to distribute oxygenand nutrients, especially glucose. Small scale fermentation refersgenerally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J. Biol. Chem. 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

c) Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the antibody products of the invention.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized can be a column comprising a glass or silica surface, ora controlled pore glass column or a silicic acid column. In someapplications, the column is coated with a reagent, such as glycerol, topossibly prevent nonspecific adherence of contaminants.

As the first step of purification, a preparation derived from the cellculture as described above can be applied onto a Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase would then be washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells:

A vector for use in a eukaryotic host cell generally includes one ormore of the following non-limiting components: a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence.

a) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected may be one that is recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such a precursor region is ligated in readingframe to DNA encoding the antibody.

b) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

c) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, primate metallothionein genes, adenosine deaminase, ornithinedecarboxylase, etc.

For example, in some embodiments, cells transformed with the DHFRselection gene are first identified by culturing all of thetransformants in a culture medium that contains methotrexate (Mtx), acompetitive antagonist of DHFR. In some embodiments, an appropriate hostcell when wild-type DHFR is employed is the Chinese hamster ovary (CHO)cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199. Host cellsmay include NSO, CHOK1, CHOK1SV or derivatives, including cell linesdeficient in glutamine synthetase (GS). Methods for the use of GS as aselectable marker for mammalian cells are described in U.S. Pat. Nos.5,122,464 and 5,891,693.

d) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding a polypeptide of interest (e.g., an antibody). Promotersequences are known for eukaryotes. For example, virtually alleukaryotic genes have an AT-rich region located approximately 25 to 30bases upstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. In certain embodiments, any or all of these sequences may besuitably inserted into eukaryotic expression vectors.

Transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, provided such promoters arecompatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982), describingexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

e) Enhancer Element Component

Transcription of DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the human cytomegalovirus early promoter enhancer, the mousecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. See alsoYaniv, Nature 297:17-18 (1982) describing enhancer elements foractivation of eukaryotic promoters. The enhancer may be spliced into thevector at a position 5′ or 3′ to the antibody polypeptide-encodingsequence, but is generally located at a site 5′ from the promoter.

f) Transcription Termination Component

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

g) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; CHOK1 cells, CHOK1SVcells or derivatives and a human hepatoma line (Hep G2).

Host cells are transformed with the above-described-expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

h) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othersupplements may also be included at appropriate concentrations thatwould be known to those skilled in the art. The culture conditions, suchas temperature, pH, and the like, are those previously used with thehost cell selected for expression, and will be apparent to theordinarily skilled artisan.

i) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, may be removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems may be firstconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis, and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing a convenient technique. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc domain that is present in the antibody. Protein A can be used topurify antibodies that are based on human γ1, γ2, or γ4 heavy chains(Lindmark et al., J. Immunol. Methods 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached may be agarose, but other matrices are available. Mechanicallystable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to furtherpurification, for example, by low pH hydrophobic interactionchromatography using an elution buffer at a pH between about 2.5-4.5,performed at low salt concentrations (e.g., from about 0-0.25M salt).

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical use are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

Production of Non-Fucosylated Antibodies

Provided herein are methods for preparing antibodies with a reduceddegree of fucosylation. For example, methods contemplated hereininclude, but are not limited to, use of cell lines deficient in proteinfucosylation (e.g., Lec13 CHO cells, alpha-1,6-fucosyltransferase geneknockout CHO cells, cells overexpressingβ1,4-N-acetylglycosminyltransferase III and further overexpressing Golgiμ-mannosidase II, etc,), and addition of a fucose analog(s) in a cellculture medium used for the production of the antibodies. See Ripka etal. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; WO 2004/056312 A1: Yamane-Ohnuki et al,Biotech. Bioeng. 87: 614 (2004); and U.S. Pat. No. 8,574,907. Additionaltechniques for reducing the fucose content of antibodies include Glymaxxtechnology described in U.S. Patent Application Publication No.2012/0214975. Additional techniques for reducing the fucose content ofantibodies also include the addition of one or more glycosidaseinhibitors in a cell culture medium used for the production of theantibodies. Glycosidase inhibitors include α-glucosidase I,α-glucosidase II, and α-mannosidase I. In some embodiments, theglycosidase inhibitor is an inhibitor of α-mannosidase I (e.g.,kifunensine).

As used herein, “core fucosylation” refers to addition of fucose(“fucosylation”) to N-acetylglucosamine (“GlcNAc”) at the reducingterminal of an N-linked glycan. Also provided are antibodies produced bysuch methods and compositions thereof.

In some embodiments, fucosylation of complex N-glycoside-linked sugarchains bound to the Fc region (or domain) is reduced. As used herein, a“complex N-glycoside-linked sugar chain” is typically bound toasparagine 297 (according to the number of Kabat), although a complexN-glycoside linked sugar chain can also be linked to other asparagineresidues. A “complex N-glycoside-linked sugar chain” excludes a highmannose type of sugar chain, in which only mannose is incorporated atthe non-reducing terminal of the core structure, but includes 1) acomplex type, in which the non-reducing terminal side of the corestructure has one or more branches of galactose-N-acetylglucosamine(also referred to as “gal-GlcNAc”) and the non-reducing terminal side ofGal-GlcNAc optionally has a sialic acid, bisecting N-acetylglucosamineor the like; or 2) a hybrid type, in which the non-reducing terminalside of the core structure has both branches of the high mannoseN-glycoside-linked sugar chain and complex N-glycoside-linked sugarchain.

In some embodiments, the “complex N-glycoside-linked sugar chain”includes a complex type in which the non-reducing terminal side of thecore structure has zero, one or more branches ofgalactose-N-acetylglucosamine (also referred to as “gal-GlcNAc”) and thenon-reducing terminal side of Gal-GlcNAc optionally further has astructure such as a sialic acid, bisecting N-acetylglucosamine or thelike.

According to the present methods, typically only a minor amount offucose is incorporated into the complex N-glycoside-linked sugarchain(s). For example, in various embodiments, less than about 60%, lessthan about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,or less than about 1% of the antibody has core fucosylation by fucose ina composition. In some embodiments, substantially none (i.e., less thanabout 0.5%) of the antibody has core fucosylation by fucose in acomposition. In some embodiments, more than about 40%, more than about50%, more than about 60%, more than about 70%, more than about 80%, morethan about 90%, more than about 91%, more than about 92%, more thanabout 93%, more than about 94%, more than about 95%, more than about96%, more than about 97%, more than about 98%, or more than about 99% ofthe antibody is nonfucosylated in a composition.

In some embodiments, provided herein is an antibody whereinsubstantially none (i.e., less than about 0.5%) of theN-glycoside-linked carbohydrate chains contain a fucose residue. In someembodiments, provided herein is an antibody wherein at least one or twoof the heavy chains of the antibody is non-fucosylated.

As described above, a variety of mammalian host-expression vectorsystems can be utilized to express an antibody. In some embodiments, theculture media is not supplemented with fucose. in some embodiments, aneffective amount of a fucose analog is added to the culture media. Inthis context, an “effective amount” refers to an amount of the analogthat is sufficient to decrease fucose incorporation into a complexN-glycoside-linked sugar chain of an antibody by at least about 10%, atleast about 20%, at least about 30%, at least about 40% or at leastabout 50%. In some embodiments, antibodies produced by the instantmethods comprise at least about 10%, at least about 20%, at least about30%, at least about 40% or at least about 50% non-core fucosylatedprotein (e.g., lacking core fucosylation), as compared with antibodiesproduced from the host cells cultured in the absence of a fucose analog.

The content (e.g., the ratio) of sugar chains in which fucose is notbound to N-acetylglucosamine in the reducing end of the sugar chainversus sugar chains in which frucose is bound to N-acetylglucosamine inthe reducing end of the sugar chain can be determined, for example, asdescribed in the Examples. Other methods include hydrazinolysis orenzyme digestion (see, e.g., Biochemical Experimentation Methods 23:Method for Studying Glycoprotein Sugar Chain (Japan Scientific SocietiesPress), edited by Reiko Takahashi (1989)), fluorescence labeling orradioisotope labeling of the released sugar chain and then separatingthe labeled sugar chain by chromatography. Also, the compositions of thereleased sugar chains can be determined by analyzing the chains by theHPAEC-PAD method (see, e.g., J. Liq Chromatogr. 6:1557 (1983)). (Seegenerally U.S. Patent Application Publication No. 2004/0110282.).

IV. Compositions

In some aspects, also provided herein are compositions (e.g.,pharmaceutical composition) comprising any of the anti-Siglec-8antibodies described herein. In some aspects, provided herein is acomposition comprising an anti-Siglec-8 antibody described herein,wherein the antibody comprises a Fc region and N-glycoside-linkedcarbohydrate chains linked to the Fc region, wherein less than about 50%of the N-glycoside-linked carbohydrate chains contain a fucose residue.In some aspects, provided herein is a composition comprising ananti-Siglec-8 antibody described herein, wherein the antibody comprisesa Fc region and N-glycoside-linked carbohydrate chains linked to the Fcregion, wherein substantially none of the N-glycoside-linkedcarbohydrate chains contain a fucose residue.

Therapeutic formulations are prepared for storage by mixing the activeingredient having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington: The Science and Practice of Pharmacy, 20th Ed., LippincottWilliams & Wiklins, Pub., Gennaro Ed., Philadelphia, Pa. 2000).Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers, antioxidants including ascorbic acid, methionine, Vitamin E,sodium metabisulfite; preservatives, isotonicifiers, stabilizers, metalcomplexes (e.g. Zn-protein complexes); chelating agents such as EDTAand/or non-ionic surfactants.

Buffers can be used to control the pH in a range which optimizes thetherapeutic effectiveness, especially if stability is pH dependent.Buffers can be present at concentrations ranging from about 50 mM toabout 250 mM. Suitable buffering agents for use with the presentinvention include both organic and inorganic acids and salts thereof.For example, citrate, phosphate, succinate, tartrate, fumarate,gluconate, oxalate, lactate, acetate. Additionally, buffers may becomprised of histidine and trimethylamine salts such as Tris.

Preservatives can be added to prevent microbial growth, and aretypically present in a range from about 0.2%-1.0% (w/v). Suitablepreservatives for use with the present invention includeoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium halides (e.g., chloride, bromide, iodide), benzethoniumchloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabenssuch as methyl or propyl paraben; catechol; resorcinol; cyclohexanol,3-pentanol, and m-cresol.

Tonicity agents, sometimes known as “stabilizers” can be present toadjust or maintain the tonicity of liquid in a composition. When usedwith large, charged biomolecules such as proteins and antibodies, theyare often termed “stabilizers” because they can interact with thecharged groups of the amino acid side chains, thereby lessening thepotential for inter and intra-molecular interactions. Tonicity agentscan be present in any amount between about 0.1% to about 25% by weightor between about 1 to about 5% by weight, taking into account therelative amounts of the other ingredients. In some embodiments, tonicityagents include polyhydric sugar alcohols, trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol.

Additional excipients include agents which can serve as one or more ofthe following: (1) bulking agents, (2) solubility enhancers, (3)stabilizers and (4) and agents preventing denaturation or adherence tothe container wall. Such excipients include: polyhydric sugar alcohols(enumerated above); amino acids such as alanine, glycine, glutamine,asparagine, histidine, arginine, lysine, ornithine, leucine,2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugaralcohols such as sucrose, lactose, lactitol, trehalose, stachyose,mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol,galactose, galactitol, glycerol, cyclitols (e.g., inositol),polyethylene glycol; sulfur containing reducing agents, such as urea,glutathione, thioctic acid, sodium thioglycolate, thioglycerol,a-monothioglycerol and sodium thio sulfate; low molecular weightproteins such as human serum albumin, bovine serum albumin, gelatin orother immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose,glucose; disaccharides (e.g., lactose, maltose, sucrose); trisaccharidessuch as raffinose; and polysaccharides such as dextrin or dextran.

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe present to help solubilize the therapeutic agent as well as toprotect the therapeutic protein against agitation-induced aggregation,which also permits the formulation to be exposed to shear surface stresswithout causing denaturation of the active therapeutic protein orantibody. Non-ionic surfactants are present in a range of about 0.05mg/ml to about 1.0 mg/ml or about 0.07 mg/ml to about 0.2 mg/ml. In someembodiments, non-ionic surfactants are present in a range of about0.001% to about 0.1% w/v or about 0.01% to about 0.1% w/v or about 0.01%to about 0.025% w/v.

Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80,etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®,polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.),lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenatedcastor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acidester, methyl celluose and carboxymethyl cellulose. Anionic detergentsthat can be used include sodium lauryl sulfate, dioctyle sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents includebenzalkonium chloride or benzethonium chloride.

In order for the formulations to be used for in vivo administration,they must be sterile. The formulation may be rendered sterile byfiltration through sterile filtration membranes. The therapeuticcompositions herein generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The route of administration is in accordance with known and acceptedmethods, such as by single or multiple bolus or infusion over a longperiod of time in a suitable manner, e.g., injection or infusion bysubcutaneous, intravenous, intraperitoneal, intramuscular,intraarterial, intralesional or intraarticular routes, topicaladministration, inhalation or by sustained release or extended-releasemeans.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition may comprise a cytotoxicagent, cytokine or growth inhibitory agent. Such molecules are suitablypresent in combination in amounts that are effective for the purposeintended.

V. Methods of Treatment

Provided herein are methods for treating or preventing a diseasemediated by cells expressing Siglec-8 in a subject comprisingadministering to the subject an effective amount of an anti-Siglec-8antibody described herein (e.g., a humanized anti-Siglec-8 antibody) orcompositions thereof. In some embodiments, the subject (e.g., a humanpatient) has been diagnosed with an eosinophil-mediated disorder or isat risk of developing the eosinophil-mediated disorder. In someembodiments, the subject (e.g., a human patient) has been diagnosed witha mast cell-mediated disorder or is at risk of developing the mastcell-mediated disorder. In some embodiments, the subject has aneosinophil-mediated disorder or a mast cell-mediated disorder.

Provided herein are methods of depletion or reduction of eosinophilscomprising administering to a subject an effective amount of ananti-Siglec-8 antibody described herein (e.g., a humanized anti-Siglec-8antibody). In some embodiments, the depletion or reduction ofeosinophils is measured by comparing the eosinophil population number ina sample (e.g., a tissue sample) from a subject after treatment with theantibody to the eosinophil population number in a sample from a subjectbefore treatment with the antibody. In some embodiments, the depletionor reduction of eosinophils is measured by comparing the eosinophilpopulation number in a sample (e.g., a tissue sample) from a subjectafter treatment with the antibody to the eosinophil population number ina sample from another subject without the antibody treatment or averageeosinophil population number in samples from subjects without theantibody treatment. In some embodiments, the sample is a tissue sample(e.g., a lung sample, a nasal polyposis sample, etc.). In someembodiments, depletion of reduction of eosinophils is due to apoptosisof activated eosinophils. Eosinophils can be activated or sensitized bycytokines or hormones such as, but not limited to, IL-5, GM-CSF, IL-33,IFN-γ, TNF-α, and leptin. In some embodiments, depletion of reduction ofeosinophils is due to apoptosis of resting eosinophils. In someembodiments, depletion of reduction of eosinophils is due toantibody-dependent cell-mediated cytotoxicity (ADCC). In someembodiments, the eosinophil production of inflammatory mediators isprevented or reduced. Exemplary inflammatory mediators include, but arenot limited to, reactive oxygen species, granule proteins (e.g.,eosinophil cationic protein, major basic protein, eosinophil-derivedneurotoxin, eosinophil peroxidase, etc.), lipid mediators (e.g., PAF,PGE1, PGE2, etc.) , enzymes (e.g., elastase), growth factors (e.g.,VEGF, PDGF, TGF-α, TGF-β, etc.), chemokines (e.g., RANTES, MCP-1, MCP-3,MCP4, eotaxin, etc.) and cytokines (e.g., IL-3, IL-5, IL-10, IL-13,IL-15, IL-33, TNF-α, etc.).

Provided herein are also methods of depletion or reduction of mast cellscomprising administering to a subject an effective amount of ananti-Siglec-8 antibody described herein (e.g., a humanized anti-Siglec-8antibody). In some embodiments, the depletion or reduction of mast cellsis measured by comparing the mast cell population number in a sample(e.g., a tissue sample or a biological fluid sample) from a subjectafter treatment with the antibody to the mast cell population number ina sample from a subject before treatment with the antibody. In someembodiments, the depletion or reduction of mast cells is measured bycomparing the mast cell population number in a sample (e.g., a tissuesample or a biological fluid sample) from a subject after treatment withthe antibody to the mast cell population number in a sample from anothersubject without the antibody treatment or average mast cell populationnumber in samples from subjects without the antibody treatment. In someembodiments, the sample is a tissue sample (e.g., a skin sample, a lungsample, a bone marrow sample, a nasal polyposis sample, etc.). In someembodiments, the sample is a biological fluid sample (e.g., a bloodsample, a bronchoalveolar lavage sample, and a nasal lavage sample). Insome embodiments, depletion of reduction of mast cells is due toantibody-dependent cell-mediated cytotoxicity (ADCC). In someembodiments, depletion or reduction of mast cells is the reduction orprevention of preformed or newly formed inflammatory mediators producedfrom mast cells. Exemplary inflammatory mediators include, but are notlimited to, histamine, N-methyl histamine, enzymes (e.g., tryptase,chymase, cathespin G, carboxypeptidase, etc.), lipid mediators (e.g.,prostaglandin D2, prostaglandin E2, leukotriene B4, leukotriene C4,platelet-activating factor, 11-beta-prostaglandin F2, etc.), chemokines(e.g., CCL2, CCL3, CCL4, CCL11 (i.e., eotaxin), CXCL1, CXCL2, CXCL3,CXCL10, etc.), and cytokines (e.g., IL-3, IL-4, IL-5, IL-15, IL-33,GM-CSF, TNF, etc.).

Provided herein are also methods of depleting mast cells expressingSiglec-8 in a subject comprising administering to the subject aneffective amount of an anti-Siglec-8 antibody described herein (e.g., ahumanized anti-Siglec-8 antibody), wherein the anti-Siglec-8 antibodykills mast cells expressing Siglec-8 by ADCC activity. In someembodiments, the anti-Siglec-8 antibody depletes at least about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90% or about 100% of the mast cells expressing Siglec-8 in a sampleobtained from the subject as compared to a baseline level beforetreatment. In some embodiments, the anti-Siglec-8 antibody depletes atleast about 20% of the mast cells expressing Siglec-8 in a sampleobtained from the subject as compared to a baseline level beforetreatment. In some embodiments, the depletion or killing of mast cellsis measured by comparing the mast cell population number in a sample(e.g., a tissue sample or a biological fluid sample) from a subjectafter treatment with the antibody to the mast cell population number ina sample from a subject before treatment with the antibody. In someembodiments, the depletion or killing of mast cells is measured bycomparing the mast cell population number in a sample (e.g., a tissuesample or a biological fluid sample) from a subject after treatment withthe antibody to the mast cell population number in a sample from anothersubject without the antibody treatment or average mast cell populationnumber in samples from subjects without the antibody treatment. In someembodiments, the sample is a tissue sample (e.g., a skin sample, a lungsample, a bone marrow sample, a nasal polyposis sample, etc.). In someembodiments, the sample is a biological fluid sample (e.g., a bloodsample, a bronchoalveolar lavage sample, and a nasal lavage sample). Insome embodiments, the anti-Siglec-8 antibody has been engineered toimprove ADCC activity. In some embodiments, the anti-Siglec-8 antibodycomprises at least one amino acid substitution in the Fc region thatimproves ADCC activity. In some embodiments, at least one or two of theheavy chains of the antibody is non-fucosylated. In some embodiments,depletion or killing of mast cells is the reduction or prevention ofpreformed or newly formed inflammatory mediators produced from mastcells. Exemplary inflammatory mediators include, but are not limited to,histamine, N-methyl histamine, enzymes (e.g., tryptase, chymase,cathespin G, carboxypeptidase, etc.), lipid mediators (e.g.,prostaglandin D2, prostaglandin E2, leukotriene B4, leukotriene C4,platelet-activating factor, 11-beta-prostaglandin F2, etc.), chemokines(e.g., CCL2, CCL3, CCL4, CCL11 (i.e., eotaxin), CXCL1, CXCL2, CXCL3,CXCL10, etc.), and cytokines (e.g., IL-3, IL-4, IL-5, IL-13, IL-15,IL-33, GM-CSF, TNF, etc.).

Also provided herein are methods of inhibiting mast cell-mediatedactivity comprising administering to a subject an effective amount of ananti-Siglec-8 antibody described herein (e.g., a humanized anti-Siglec-8antibody). In some embodiments, the inhibition of mast cell-mediatedactivity is measured by comparing the mast cell-mediated activity in asample (e.g., a tissue sample or a blood sample) from a subject aftertreatment with the antibody to the mast cell-mediated activity in asample from a subject before treatment with the antibody. In someembodiments, the inhibition of mast cell-mediated activity is measuredby comparing the mast cell-mediated activity in a sample (e.g., a tissuesample or a biological sample) from a subject after treatment with theantibody to the mast cell-mediated activity in a sample from anothersubject without the antibody treatment or average mast cell-mediatedactivity in samples from subjects without the antibody treatment. Insome embodiments, the sample is a tissue sample (e.g., a skin sample, alung sample, a bone marrow sample, a nasal polyposis sample, etc.). Insome embodiments, the sample is a biological fluid sample (e.g., a bloodsample, a bronchoalveolar lavage sample, and a nasal lavage sample). Insome embodiments, inhibition of mast cell-mediated activity is theinhibition of mast cell degranulation. In some embodiments, inhibitionof mast cell-mediated activity is the inhibition of airway smooth musclecontraction. In some embodiments, inhibition of mast cell-mediatedactivity is the inhibition of calcium flux in mast cells. In someembodiments, inhibition of mast cell-mediated activity is the inhibitionof release of preformed or newly formed inflammatory mediators from mastcells. Exemplary inflammatory mediators include, but are not limited to,histamine, N-methyl histamine, enzymes (e.g., tryptase, chymase,cathespin G, carboxypeptidase, etc.), lipid mediators (e.g.,prostaglandin D2, prostaglandin E2, leukotriene B4, leukotriene C4,platelet-activating factor, 11-beta-prostaglandin F2, etc.), chemokines(e.g., CCL2, CCL3, CCL4, CCL11 (i.e., eotaxin), CXCL1, CXCL2, CXCL3,CXCL10, etc.), and cytokines (e.g., IL-3, IL-4, IL-5, IL-13, IL-15,IL-33, GM-CSF, TNF, etc.).

For the prevention or treatment of disease, the appropriate dosage of anactive agent, will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether the agentis administered for preventive or therapeutic purposes, previoustherapy, the subject's clinical history and response to the agent, andthe discretion of the attending physician. The agent is suitablyadministered to the subject at one time or over a series of treatments.In some embodiments of the methods described herein, an interval betweenadministrations of an anti-Siglec-8 antibody described is about onemonth or longer. In some embodiments, the interval betweenadministrations is about two months, about three months, about fourmonths, about five months, about six months or longer. As used herein,an interval between administrations refers to the time period betweenone administration of the antibody and the next administration of theantibody. As used herein, an interval of about one month includes fourweeks. Accordingly, in some embodiments, the interval betweenadministrations is about four weeks, about eight weeks, about twelveweeks, about sixteen weeks, about twenty weeks, about twenty four weeks,or longer. In some embodiments, the treatment includes multipleadministrations of the antibody, wherein the interval betweenadministrations may vary. For example, the interval between the firstadministration and the second administration is about one month, and theintervals between the subsequent administrations are about three months.In some embodiments, the interval between the first administration andthe second administration is about one month, the interval between thesecond administration and the third administration is about two months,and the intervals between the subsequent administrations are about threemonths. In some embodiments, an anti-Siglec-8 antibody described hereinis administered at a flat dose. In some embodiments, an anti-Siglec-8antibody described herein is administered to a subject at a dosage fromabout 150 to about 450 mg per dose. In some embodiments, theanti-Siglec-8 antibody is administered to a subject at a dosage of aboutany of 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, and 450 mg perdose. In some embodiments, an anti-Siglec-8 antibody described herein isadministered to a subject at a dosage from about 0.1 mg/kg to about 10mg/kg or about 1.0 mg/kg to about 10 mg/kg. In some embodiments, ananti-Siglec-8 antibody described herein is administered to a subject ata dosage of about any of 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg,5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5mg/kg, 9.0 mg/kg, 9.5 mg/kg, or 10.0 mg/kg. Any of the dosing frequencydescribed above may be used.

A method of treatment contemplated herein is the treatment ofeosinophil-mediated disorders and/or mast cell-mediated disorders withan anti-Siglec-8 antibody described herein. Eosinophil-mediateddisorders include a disorder or disease associated with eosinophilmigration, chemotaxis, generation, or granulation. Similarly, mastcell-mediated disorders include a disorder or disease associated withmast cell migration, chemotaxis, generation, or granulation.Eosinophil-mediated disorders and/or mast cell-mediated disorders thatare treatable with the formulations of this present invention includeasthma, allergic rhinitis, nasal polyposis, atopic dermatitis, chronicurticaria (e.g., chronic idiopathic urticaria and chronic spontaneousurticaria), mastocytosis, eosinophilic leukemia, and hypereosinophilicsyndrome. Eosinophil-mediated disorders and/or mast cell-mediateddisorders that are treatable with the formulations of this presentinvention also include pauci granulocytic asthma, acute or chronicairway hypersensitivity, eosinophilic esophagitis, Churg-Strausssyndrome, inflammation associated with a cytokine, inflammationassociated with cells expressing Siglec-8, malignancy associated withcells expressing Siglec-8, physical urticaria, cold urticaria,pressure-urticaria, bullous pemphigoid, food allergy, and allergicbronchopulmonary aspergillosis (ABPA).

In some embodiments of the methods herein, an anti-Siglec-8 antibodyprovided herein inhibits one or more symptoms of an allergic reaction.In some embodiments, the allergic reaction is a Type I hypersensitivityreaction.

Allergic rhinitis, also known as allergic rhinoconjunctivitis or hayfever, is the most common manifestation of an atopic reaction to inhaledallergens, the severity and duration of which is often correlative withthe intensity and length of exposure to the allergen. It is a chronicdisease, which may first appear at any age, but the onset is usuallyduring childhood or adolescence. A typical attack consists of profusewatery rhinorrhea, paroxysmal sneezing, nasal obstruction and itching ofthe nose and palate. Postnasal mucus drainage also causes sore throat,throat clearing and cough. There can also be symptoms of allergicblepharoconjunctivitis, with intense itching of the conjunctivae andeyelids, redness, tearing, and photophobia. Severe attacks are oftenaccompanied by systemic malaise, weakness, fatigue, and sometimes,muscle soreness after intense periods of sneezing.

Asthma, also known as reversible obstructive airway disease, ischaracterized by hyperresponsiveness of the tracheobronchial tree torespiratory irritants and bronchoconstrictor chemicals, producingattacks of wheezing, dyspnea, chest tightness, and cough that arereversible spontaneously or with treatment. It is a chronic diseaseinvolving the entire airway, but varies in severity from occasional mildtransient episodes to severe, chronic, life-threatening bronchialobstruction. Physical signs of an asthma attack include tachypnea,audible wheezing, and use of the accessory muscles of respiration. Rapidpulse and elevated blood pressure are also typically present, as areelevated levels of eosinophils in the peripheral blood and nasalsecretions. Asthma and atopy may coexist, but only about half ofasthmatics are also atopic, and an even smaller percentage of atopicpatients also have asthma. However, atopy and asthma are not entirelyindependent in that asthma occurs more frequently among atopic thanamongst nonatopic individuals, especially during childhood. Asthma hasfurther been historically broken down into two subgroups, extrinsicasthma and intrinsic asthma. In addition, asthma involves chronicinflammation of the airways, acute exacerbations varying in frequencybetween different patients and in response to environmental triggers. Insevere cases, chronic remodeling of the airways may occur.

Extrinsic asthma, also known as allergic, atopic or immunologic asthma,is descriptive of patients that generally develop asthma early in life,usually during infancy or childhood. Other manifestations of atopy,including eczema or allergic rhinitis often coexist. Asthmatic attackscan occur during pollen seasons, in the presence of animals, or onexposure to house dust, feather pillows, or other allergens. Skin testsshow positive wheal-and-flare reactions to the causative allergens.Interestingly, serum total IgE concentrations are frequently elevated,but are sometimes normal. Intrinsic asthma, also known as nonallergic oridopathic asthma, typically first occurs during adult life, after anapparent respiratory infection. Symptoms include chronic or recurrentbronchial obstruction unrelated to pollen seasons or exposure to otherallergens. Skin tests are negative to the usual atopic allergens, serumIgE concentration is normal. Additional symptoms include sputum bloodand eosinophilia. For purposes of this patent application,“eosinophil-mediated disorders” includes both allergic and non-allergicasthma. In some embodiments, a subject with an eosinophil-mediateddisorder(s) and/or mast cell-mediated disorder(s) is suffering fromasthma that is not adequately controlled by an inhaled corticosteroid, ashort acting β2 agonist, a long acting β2 agonist, or a combinationthereof

Atopic dermatitis, also known as eczema, neurodermatitis, atopic eczemaor Besnier's prurigo, is common chronic skin disorder specific to asubset of patients with the familial and immunologic features of atopy.The essential feature is a pruritic dermal inflammatory response, whichinduces a characteristic symmetrically distributed skin eruption withpredilection for certain sites. While atopic dermatitis is classified asa cutaneous form of atopy because it is associated with allergicrhinitis and asthma and high IgE levels, the severity of the dermatitis,however, does not always correlate with exposure to allergens on skintesting, and desensitization (unlike other allergic diseases) is noteffective treatment. While high serum IgE is confirmatory of a diagnosisof allergic asthma, normal levels do not preclude it. Onset of thedisease can occur at any age, and lesions begin acutely witherythematous edematous papule or plaque with scaling. Itching leads toweeping and crusting, then to chronic lichenification. On the cellularlevel, acute lesion is edemous and the dermis is infiltrated withmononuclear cells, CD4 lymphocytes. Neutrophils, eosinophils, plasmacells and basophils are rare, but degranulated mast cells are present.Chronic lesions feature epidermal hyperplasia, hyperkeratosis andparakeratosis, and the dermis is infiltrated with mononuclear cells,Langerhans' cells and mast cells. There may also be focal areas offibrosis, including involvement of the perineurium of small nerves.

Urticaria and angioedema refers to the physical swelling, erythema anditching resulting from histamine stimulated receptor in superficialcutaneous blood vessels, and is the hallmark cutaneous feature ofsystemic anaphylaxis. Systemic anaphylaxis is the occurrence of anIgE-mediated reaction simultaneously in multiple organs resulting fromdrug, insect venom or food. It is caused suddenly by allergen induced,mast cell loaded IgE, resulting in profound and life-threateningalteration in the functioning of various vital organs. Vascularcollapse, acute airway obstruction, cutaneous vasodilation and edema,and gastrointestinal and genitourinary muscle spasm occur almostsimultaneously, although not always to the same degree. The pathology ofanaphylaxis includes angioedema and hyperinflated lungs, with mucousplugging of airways and focal atelectasis. On a cellular level, thelungs appear similarly as during an acute asthma attack, withhypersecretion of bronchial submucosal glands, mucosal and submucosaledema, peribronchial vascular congestion and eosinophilia in thebronchial walls. Pulmonary edema and hemorrhage may be present.Bronchial muscle spasm, hyperinflation, and even rupture of alveoli mayalso be present. Important features of human anaphylaxis include edema,vascular congestion, and eosinophilia in the lamina propria of thelarynx, trachea, epiglottis and hypopharynx. Exposure to the allergenmay be through ingestion, injection, inhalation or contact with skin ormucous membrane. The reaction begins within seconds or minutes afterexposure to the allergen. There may be an initial fright or sense ofimpending doom, followed rapidly by symptoms in one or more target organsystems: cardiovascular, respiratory, cutaneous or gastrointestinal. Theallergens responsible for anaphylaxis differ from those commonlyassociated with atopy. Foods, drugs, insect venoms or latex are thecommon sources. Food allergens includes those found in crustaceans,mollusks (e.g., lobster, shrimp, crab), fish, legumes (e.g., peanuts,peas, beans, licorice), seeds (e.g. sesame, cottonseed, caraway,mustard, flaxseed, sunflower), nuts, berries, egg whites, buckwheat andmilk. Drug allergens include those found in heterologous proteins andpolypeptides, polysaccharides and haptenic drugs. Insect allergensinclude Hymenoptera insects, including the honeybee, yellow jacket,hornet, wasp and fire ant. While epinephrine is the typical treatmentfor anaphylaxis, antihistamine or other histamine blockers are typicallyprescribed for less severe urticaria or angioedemic reaction.

Nasal polyposis is a chronic inflammatory disease of the upperrespiratory tract characterized by an outgrowth of inflamed tissue intothe nasal cavity, and although the exact etiology is unknown, it isknown to have prevalence between 1 to 5% of adults (Settipane G A:Epidemiology of nasal polyps. Allergy Asthma Proc, 1996, 17:231-236).Nasal polyposis typically presents in males 20 years of age or older andcauses nasal obstruction, hyposmia, and recurrent infections with asignificantly higher impact to quality of life than perennial allergicrhinitis (Li et al., Characterizing T-Cell Phenotypes in Nasal Polyposisin Chinese Patients, J Investig Allergol Clin Immunol, 2009; Vol.19(4):276-282). Up to one third of all patients with nasal polyposis arereported to have asthma however only 7% of asthma patients have nasalpolyposis. Predominate cell types implicated in nasal polyposis includeeosinophils and mast cells.

VI. Articles of Manufacture or Kits

In another aspect, an article of manufacture or kit is provided whichcomprises an anti-Siglec-8 antibody described herein. The article ofmanufacture or kit may further comprise instructions for use of theantibody in the methods of the invention. Thus, in certain embodiments,the article of manufacture or kit comprises instructions for the use ofan anti-Siglec-8 antibody in methods for treating or preventing aneosinophil-mediated disorder and/or mast cell-mediated disorder in anindividual comprising administering to the individual an effectiveamount of an anti-Siglec-8 antibody. In certain embodiments, theindividual is a human. In some embodiments, the individual has a diseaseselected from the group consisting of asthma, allergic rhinitis, nasalpolyposis, atopic dermatitis, chronic urticaria, mastocytosis,eosinophilic leukemia, and hypereosinophilic syndrome. In certainembodiments, the individual has a disease selected from the groupconsisting of pauci granulocytic asthma, acute or chronic airwayhypersensitivity, eosinophilic esophagitis, Churg-Strauss syndrome,inflammation associated with a cytokine, inflammation associated withcells expressing Siglec-8, malignancy associated with cells expressingSiglec-8, physical urticaria, cold urticaria, pressure-urticaria,bullous pemphigoid, food allergy, and allergic bronchopulmonaryaspergillosis (ABPA).

The article of manufacture or kit may further comprise a container.Suitable containers include, for example, bottles, vials (e.g., dualchamber vials), syringes (such as single or dual chamber syringes) andtest tubes. The container may be formed from a variety of materials suchas glass or plastic. The container holds the formulation.

The article of manufacture or kit may further comprise a label or apackage insert, which is on or associated with the container, mayindicate directions for reconstitution and/or use of the formulation.The label or package insert may further indicate that the formulation isuseful or intended for subcutaneous, intravenous, or other modes ofadministration for treating or preventing an eosinophil-mediateddisorder and/or mast cell-mediated disorder in an individual. Thecontainer holding the formulation may be a single-use vial or amulti-use vial, which allows for repeat administrations of thereconstituted formulation. The article of manufacture or kit may furthercomprise a second container comprising a suitable diluent. The articleof manufacture or kit may further include other materials desirable froma commercial, therapeutic, and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

In a specific embodiment, the present invention provides kits for asingle dose-administration unit. Such kits comprise a container of anaqueous formulation of therapeutic antibody, including both single ormulti-chambered pre-filled syringes. Exemplary pre-filled syringes areavailable from Vetter GmbH, Ravensburg, Germany.

The article of manufacture or kit herein optionally further comprises acontainer comprising a second medicament, wherein the anti-Siglec-8antibody is a first medicament, and which article or kit furthercomprises instructions on the label or package insert for treating thesubject with the second medicament, in an effective amount. An exemplarysecond medicament may be an anti-IgE antibody, an antihistamine, abronchodilator, a glucocorticoid, an NSAID, a decongestant, a coughsuppressant, an analgesic, a TNF-antagonist, an integrin antagonist, animmunosuppressive agent, an IL-4 antagonist, an IL-13 antagonist, a dualIL-4/IL-13 antagonist, a DMARD, an antibody that binds to a B-cellsurface marker, and/or a BAFF antagonist.

In another embodiment, provided herein is an article of manufacture orkit comprising the formulations described herein for administration inan auto-injector device. An auto-injector can be described as aninjection device that upon activation, will deliver its contents withoutadditional necessary action from the patient or administrator. They areparticularly suited for self-medication of therapeutic formulations whenthe delivery rate must be constant and the time of delivery is greaterthan a few moments.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

EXAMPLES

Siglecs (sialic acid-binding, immunoglobulin-like lectins) aresingle-pass transmembrane cell surface proteins found predominantly onleukocytes. Siglec-8, a member of the Siglec family, was firstdiscovered as part of efforts to identify novel human eosinophilproteins. In addition to expression with human eosinophils, it is alsoexpressed by mast cells. Siglec-8 recognizes a sulfated glycan,6′-sulfo-sialyl Lewis X, and contains an intracellular immunoreceptortyrosine-based inhibitory motif (ITIM) domain shown to inhibit mast cellfunction. Murine antibodies to Siglec-8, 2E2 and 2C4 antibody aredescribed in U.S. Pat. Nos. 8,207,305; 8,197,811, 7,871,612, and7,557,191. The amino acid sequence of the heavy chain variable domainand light chain variable domain of the mouse anti-Siglec-8 2C4 antibodycan be found, for example, in U.S. Pat. No. 8,207,305 as SEQ ID NO:2 andSEQ ID NO:4, respectively.

Example 1: Generation and Characterization of Chimeric Anti-Siglec-8Antibodies

Chimeric antibodies were generated from the mouse 2E2 antibody and mouse2C4 antibody, and were subsequently analyzed for binding activity tohuman Siglec-8.

Methods and Results

Generation of Chimeric 2E2 Antibody (ch2E2) and Chimeric 2C4 Antibody(ch2C4)

For generation of a chimeric 2E2 antibody (ch2E2), snap-frozen mousehybridoma 2E2 cell lysates were processed using the RNeasy Mini kit(Qiagen) to isolate total RNA according to the manufacturer's protocol.A 3 μg sample of isolated RNA was reverse-transcribed to produce 2E2cDNA using the 1st strand cDNA synthesis kit (GE Life Sciences)according to the manufacturer's protocol. 2E2 cDNA was subsequentlyamplified by PCR using Phusion Flash High-Fidelity PCR Master Mix(Thermo Scientific) and the sequences in the PCR reactions wereconfirmed. The immunoglobulin heavy chain variable region (V_(H)) cDNAand kappa light chain variable region (VL) were PCR-amplified using thePhusion High-Fidelity PCR Master Mix. The result of each PCR reactionwas a single amplification product that was purified using the QIAquickPCR Purification kit (Qiagen) according to the manufacturer's protocoland the sequence for each immunoglobulin chain was obtained. Theconsensus sequence of the kappa light chain variable region wasdesignated 2E2 VK and the consensus sequence of the heavy light chainvariable region was designated 2E2 VH. The 2E2 VK protein sequence wasidentical to that of the mouse 2C4 IgG1 antibody (see Kikly et al., J.Allergy Clin Immunol., 2000; 105:1093-1100) except for the firstresidue, where Gln was replaced by a Glu while the 2E2 VH proteinsequence was completely identical to that of 2C4.

Construction of chimeric expression vectors entailed cloning theamplified variable regions into IgG/kappa constant region vectors usingligase-independent cloning. Briefly, the pCMV vectors were digested withBfuA1 (BsPM1), the digested vector was purified by gel electrophoresis,and compatible overhangs in the vector were generated by incubation withT4 DNA polymerase and 100 mM dATP. For the insert, antibody sequenceswere amplified by PCR from 2E2 cDNA with primers containing the 3′ endof the leader sequence, i.e., forward primer, or the beginning of theconstant region (IgG1 or kappa), i.e., reverse primer, followed by thebeginning of the variable region (in each direction). The amplifiedinserts was purified using a PCR purification kit (Qiagen) andcomplementary overhangs were generated in the insert by incubation withT4 DNA polymerase and 100 mM dTTP. Vector and inserts were incubated atroom temperature and used to transform chemically-competent TOP10bacteria (Invitrogen) that were subsequently plated on culture platescontaining kanamycin. Several clones were isolated and colonies werescreened by PCR. Clones containing the correct sized PCR products wereselected, DNA was isolated using a miniprep kit (Qiagen) and the DNA wassequenced.

For generation of a chimeric 2C4 antibody (ch2C4) , the 2C4 heavy chainvariable region (VH) and kappa light chain variable region (VK) weresynthesized (GeneScript) and the same method used to clone the chimeric2E2 antibody was applied. Expi293 suspension cells (Human EmbryonicKidney cells) growing in Expi293 transfection medium (Invitrogen) andantibiotics were co-transfected with constructs encoding ch2E2 heavychain and ch2E2 kappa light chain or constructs encoding ch2C4 heavychain and ch2C4 kappa light chain (1 μg DNA each) using ExpiFectamine293 Reagent provided with the Expi293 Expression System kit (Invitrogen)according to the manufacturer's protocol. The cells were grown in 2 mlgrowth medium in six well plates for 7 days before media was harvestedand assayed for recombinant protein expression by ELISA.

Siglec-8 Binding Activity of Chimeric Antibody

Binding of recombinant human Siglec-8 extracellular domain (ECD) bychimeric 2E2 and chimeric 2C4 IgG1K antibodies was measured by ELISA.For the Siglec-8 binding assay, a 384-well SpectraPlate (Perkin Elmer)was prepared by coating with 30 μL per well of 0.4 μg/mL Siglec-8 ECDovernight at 4° C. followed by removal of the coating solution bywashing wells in wash buffer (0.1% Tween 20), and blocking with 90 μL 5%BSA/TBS solution for 2 hours at room temperature. Three-fold serialdilutions of a chimeric antibody (ch2E2 or ch2C4) or mouse antibody(m2E2 or m2C4) in 0.2% BSA/TBS (starting concentration was 5000 ng/mL)was added to each coated well. The plate was incubated for 2 hours atroom temperature and the wells were subsequently washed before additionof goat-anti-human Fc peroxidase conjugate (1:10000 dilution) oranti-mouse Fc peroxidase conjugate (1:30000 dilution) diluted in0.2%BSA/TBS solution. The plate was incubated for 45 minutes at roomtemperature, followed by washing and addition of 30 μL K-blue HRPsubstrate (SkyBio Ltd) to each well. After incubation at roomtemperature for 15 minutes, the reaction was stopped by adding 10 μL ofRed Stop solution (SkyBio Ltd) to each well. The optical density or theexperimental samples was read at 650 nm using the ELISA reader PHERAStarFS (BMG Labtech).

Both chimeric antibodies bound full Siglec-8 ECD with comparable EC₅₀values. Chimeric antibody ch2E2 bound to Siglec-8 ECD with a lower EC₅₀value as compared to mouse antibody m2E2 (Table 2).

TABLE 2 Antibody binding to human Siglec-8 ECD. m2E2 ch2E2 purifiedch2E2 ch2C4 EC50 0.1003 0.05701 0.04759 0.07411

Example 2: Generation and Characterization of Humanized Anti-Siglec-8Antibodies

The sequence of the chimeric antibody 2E2 and chimeric antibody 2C4 wasused to design humanized versions of the mouse 2E2 antibodies.

Methods and Results

Design of Humanized Anti-Siglec-8 Antibodies

The protein sequences of human and mouse immunoglobulins from theInternational Immunogenetics Database 2009 (Lefranc, M P., Nucleic AcidRes., 2003, 31(1):307-10) and the Kabat Database Release 5 of Sequencesof Proteins of Immunological Interest (last update 17 Nov. 1999) (Kabatet al., NIH National Technical Information Service, 1991, 1-3242) wereused to compile a database of human immunoglobulin sequences in Kabatalignment. The compiled database contained 10,906 VH and 2,912 VKsequences.

A homology model of mouse antibody 2C4 variable regions had beencalculated using the Modeller program (Eswar et al., Curr. Protoc.Bioinformatics, 2006, Ch. 5: Unit 5.6) included in the Discovery Studiopackage (Accelrys, Inc.). The atomic coordinates of 1a7O.pdb, 1dqd.pdband 1ORS.pdb were the highest framework identity sequence templates forthe VH, VL and backbone/interface, respectively, as determined by BasicLocal Alignment Search Tool (BLAST) analysis of the antibody pdbstructures database (Accelrys, Inc.). These templates were used togenerate 30 initial models of the framework and the lowest energy modelwas used to generate 20 loop models (with all CDR loops included), withits 5 best loop templates, using the Kabat definition to eventuallygenerate a final mouse 2C4 model.

Humanization required the identification of suitable human V regions.The Gibbs sequence analysis program (MRCT) was used to interrogate thehuman VH and VK databases with 2C4 VH and VK protein sequences usingvarious selection criteria. Using the Discovery Studio program(Accelrys), framework (FW) residues within 4 Å of the CDR residues(Kabat definition) in the final homology model of mouse antibody 2C4were identified, and designated as the “4 Å CDR envelope”. There were nohuman VH sequences sharing CDR 1, 2 and 3 length identity with 2C4 thatpossessed a high 4 Å CDR envelope and/or VCI identity to 2C4 VH.AF471521 was the next best candidate with a CDR3 size of 14 residueswith the highest identity score of key framework residues (19/24 4Aenvelope and 18/22 VCI), while the other sequences had additionaldifferences, which rendered them at lower priority. However, AF471521had 11 somatic mutations from its germline VH gene X92218. In order tomitigate the somatic mutations, any framework residue that differed fromgermline and/or was not conserved in the mouse was back mutated to humangermline sequence. Therefore six framework residues were back mutated togermline and the remaining five residues that differed from germlinewere key framework residues and were identical to the mouse sequence.

Since a suitable acceptor human framework had been identified using ahomology model of the 2C4 antibody, the synthetic protein and DNAsequence were designed by grafting the CDRs of the 2E2 antibody onto theacceptor human framework. The initial design of 2E2RHA was the graftingof CDR 1, 2 and 3 from 2E2 VH into the acceptor FW of AF471521.Intercalation of the 2E2 VH CDRs (gray shade) into the FW sequence ofAF471521 resulted in the design of 2E2RHA, the initial humanized variant(FIG. 1). Five 4 Å CDR envelope/VCI residues, at Kabat positions 24, 48,49, 67 and 68 were not conserved in 2E2RHA, and these were back-mutatedto the mouse equivalent residue, in the humanized version 2E2RHB ormutated one at a time in the following variants: sequences wereassembled in silico and designated 2E2RHA through 2E2RHG (FIG. 1).

In order to humanize the light chain, a human kappa chain was identifiedin a similar process to that of the heavy chain. AY867246 was thesequence with the highest identity to 2E2 VK in the 4 Å CDR envelope/VCIbut had six somatic mutations. AY867246 was discarded in favor ofX93721, which contained 21/25 4Å envelope and 15/17 VCI and only onesomatic mutation from the nearest germline VK gene, X01668. Theframework from X93721 was used to design the DNA and protein for thehumanized constructs. CDR 1, 2 and 3 from 2E2 VK (gray shade) weregrafted into the acceptor FW of X93721 to generate 2E2RKA (FIG. 2).There were four unmatched 4 Å CDR envelope residues, 3, 47, 58 and 71 in2E2RKA that were back-mutated to the equivalent mouse residues invariant 2E2RKB or individually in the following variants: sequences wereassembled in silico and designated 2E2RKA through 2E2RKG (FIG. 2).

Generation of Humanized Anti-Siglec-8 Antibodies

The genes for 2E2RHA and 2E2RKA were synthesized (GenScript) and codonoptimized for human sequences. The natural human framework sequencesAF471521 and X93721, heavy and light chains, respectively, and thenatural mouse CDR sequences were assembled in silico and designated2E2RHA through 2E2RHG and 2E2RKA through 2E2RKG. Using softwarealgorithms (GenScript), the sequences were optimized by silentmutagenesis to use codons preferentially utilized by human cells andsynthesized. RHA/B and RKA/B constructs were PCR amplified with specificprimers to the expression vector and insert, as described above inExample 1 for PCR amplification of the chimeric antibodies, and insertedinto IgG/kappa constant region vectors by ligase-independent cloningreactions and used to transform TOP10 bacteria as described in Example 1for generation of chimeric antibodies. RKA and RHA were subsequentlymodified by PCR mutagenesis using the QuickChange LightningSite-Directed Mutagenesis kit (Stratagene) according to themanufacture's protocol to obtain all the human antibody variants (FIG.1, FIG. 2, Table 3, Table 4, and Table 5).

TABLE 3 Amino acid sequences of variable regions of chimeric andhumanized antibody variants Antibody Name Variable Heavy Chain VariableLight Chain ch2C4 ch2C4 VH ch2C4 VK ch2E2 ch2E2 VH (SEQ ID NO: 1) ch2E2VK (SEQ ID NO: 15) cVHKA ch2E2 VH (SEQ ID NO: 1) 2E2 RKA (SEQ ID NO: 16)cVHKB ch2E2 VH (SEQ ID NO: 1) 2E2 RKB (SEQ ID NO: 17) HAcVK 2E2 RHA (SEQID NO: 2) ch2E2 VK (SEQ ID NO: 15) HBcVK 2E2 RHB (SEQ ID NO: 3) ch2E2 VK(SEQ ID NO: 15) HAKA 2E2 RHA (SEQ ID NO: 2) 2E2 RKA (SEQ ID NO: 16) HAKB2E2 RHA (SEQ ID NO: 2) 2E2 RKB (SEQ ID NO: 17) HAKC 2E2 RHA (SEQ ID NO:2) 2E2 RKC (SEQ ID NO: 18) HAKD 2E2 RHA (SEQ ID NO: 2) 2E2 RKD (SEQ IDNO: 19) HAKE 2E2 RHA (SEQ ID NO: 2) 2E2 RKE (SEQ ID NO: 20) HAKF 2E2 RHA(SEQ ID NO: 2) 2E2 RKF (SEQ ID NO: 21) HAKG 2E2 RHA (SEQ ID NO: 2) 2E2RKG (SEQ ID NO: 22) HBKA 2E2 RHB (SEQ ID NO: 3) 2E2 RKA (SEQ ID NO: 16)HBKB 2E2 RHB (SEQ ID NO: 3) 2E2 RKB (SEQ ID NO: 17) HBKC 2E2 RHB (SEQ IDNO: 3) 2E2 RKC (SEQ ID NO: 18) HBKD 2E2 RHB (SEQ ID NO: 3) 2E2 RKD (SEQID NO: 19) HBKE 2E2 RHB (SEQ ID NO: 3) 2E2 RKE (SEQ ID NO: 20) HBKF 2E2RHB (SEQ ID NO: 3) 2E2 RKF (SEQ ID NO: 21) HBKG 2E2 RHB (SEQ ID NO: 3)2E2 RKG (SEQ ID NO: 22) HCKA 2E2 RHC (SEQ ID NO: 4) 2E2 RKA (SEQ ID NO:16) HCKB 2E2 RHC (SEQ ID NO: 4) 2E2 RKB (SEQ ID NO: 17) HCKC 2E2 RHC(SEQ ID NO: 4) 2E2 RKC (SEQ ID NO: 18) HCKD 2E2 RHC (SEQ ID NO: 4) 2E2RKD (SEQ ID NO: 19) HCKE 2E2 RHC (SEQ ID NO: 4) 2E2 RKE (SEQ ID NO: 20)HCKF 2E2 RHC (SEQ ID NO: 4) 2E2 RKF (SEQ ID NO: 21) HCKG 2E2 RHC (SEQ IDNO: 4) 2E2 RKG (SEQ ID NO: 22) HDKA 2E2 RHD (SEQ ID NO: 5) 2E2 RKA (SEQID NO: 16) HDKB 2E2 RHD (SEQ ID NO: 5) 2E2 RKB (SEQ ID NO: 17) HDKC 2E2RHD (SEQ ID NO: 5) 2E2 RKC (SEQ ID NO: 18) HDKD 2E2 RHD (SEQ ID NO: 5)2E2 RKD (SEQ ID NO: 19) HDKE 2E2 RHD (SEQ ID NO: 5) 2E2 RKE (SEQ ID NO:20) HDKF 2E2 RHD (SEQ ID NO: 5) 2E2 RKF (SEQ ID NO: 21) HDKG 2E2 RHD(SEQ ID NO: 5) 2E2 RKG (SEQ ID NO: 22) HEKA 2E2 RHE (SEQ ID NO: 6) 2E2RKA (SEQ ID NO: 16) HEKB 2E2 RHE (SEQ ID NO: 6) 2E2 RKB (SEQ ID NO: 17)HEKC 2E2 RHE (SEQ ID NO: 6) 2E2 RKC (SEQ ID NO: 18) HEKD 2E2 RHE (SEQ IDNO: 6) 2E2 RKD (SEQ ID NO: 19) HEKE 2E2 RHE (SEQ ID NO: 6) 2E2 RKE (SEQID NO: 20) HEKF 2E2 RHE (SEQ ID NO: 6) 2E2 RKF (SEQ ID NO: 21) HEKG 2E2RHE (SEQ ID NO: 6) 2E2 RKG (SEQ ID NO: 22) HFKA 2E2 RHF (SEQ ID NO: 7)2E2 RKA (SEQ ID NO: 16) HFKB 2E2 RHF (SEQ ID NO: 7) 2E2 RKB (SEQ ID NO:17) HFKC 2E2 RHF (SEQ ID NO: 7) 2E2 RKC (SEQ ID NO: 18) HFKD 2E2 RHF(SEQ ID NO: 7) 2E2 RKD (SEQ ID NO: 19) HFKE 2E2 RHF (SEQ ID NO: 7) 2E2RKE (SEQ ID NO: 20) HFKF 2E2 RHF (SEQ ID NO: 7) 2E2 RKF (SEQ ID NO: 21)HFKG 2E2 RHF (SEQ ID NO: 7) 2E2 RKG (SEQ ID NO: 22) HGKA 2E2 RHG (SEQ IDNO: 8) 2E2 RKA (SEQ ID NO: 16) HGKB 2E2 RHG (SEQ ID NO: 8) 2E2 RKB (SEQID NO: 17) HGKC 2E2 RHG (SEQ ID NO: 8) 2E2 RKC (SEQ ID NO: 18) HGKD 2E2RHG (SEQ ID NO: 8) 2E2 RKD (SEQ ID NO: 19) HGKE 2E2 RHG (SEQ ID NO: 8)2E2 RKE (SEQ ID NO: 20) HGKF 2E2 RHG (SEQ ID NO: 8) 2E2 RKF (SEQ ID NO:21) HGHG 2E2 RHG (SEQ ID NO: 8) 2E2 RKG (SEQ ID NO: 22) HA2KA 2E2 RHA2(SEQ ID NO: 9) 2E2 RKA (SEQ ID NO: 16) HA2KB 2E2 RHA2 (SEQ ID NO: 9) 2E2RKB (SEQ ID NO: 17) HB2KA 2E2 RHB2 (SEQ ID NO: 10) 2E2 RKA (SEQ ID NO:16) HB2KB 2E2 RHB2 (SEQ ID NO: 10) 2E2 RKB (SEQ ID NO: 17) HA2KF 2E2RHA2 (SEQ ID NO: 9) 2E2 RKF (SEQ ID NO: 21) HB2KF 2E2 RHB2 (SEQ ID NO:10) 2E2 RKF (SEQ ID NO: 21) HA2KC 2E2 RHA2 (SEQ ID NO: 9) 2E2 RKC (SEQID NO: 18) HA2KD 2E2 RHA2 (SEQ ID NO: 9) 2E2 RKD (SEQ ID NO: 19) HA2KE2E2 RHA2 (SEQ ID NO: 9) 2E2 RKE (SEQ ID NO: 20) HA2KF 2E2 RHA2 (SEQ IDNO: 9) 2E2 RKF (SEQ ID NO: 21) HA2KG 2E2 RHA2 (SEQ ID NO: 9) 2E2 RKG(SEQ ID NO: 22) HB2KC 2E2 RHB2 (SEQ ID NO: 10) 2E2 RKC (SEQ ID NO: 18)HB2KD 2E2 RHB2 (SEQ ID NO: 10) 2E2 RKD (SEQ ID NO: 19) HB2KE 2E2 RHB2(SEQ ID NO: 10) 2E2 RKE (SEQ ID NO: 20) HA2KFmut 2E2 RHA2 (SEQ ID NO: 9)2E2 RKF F-Y mut (SEQ ID NO: 24) HB2KFmut 2E2 RHB2 (SEQ ID NO: 10) 2E2RKF F-Y mut (SEQ ID NO: 24) HEKAmut 2E2 RHE (SEQ ID NO: 6) 2E2 RKA F-Ymut (SEQ ID NO: 23) HEKFmut 2E2 RHE (SEQ ID NO: 6) 2E2 RKF F-Y mut (SEQID NO: 24) HAKFmut 2E2 RHA (SEQ ID NO: 2) 2E2 RKF F-Y mut (SEQ ID NO:24) HBKFmut 2E2 RHB (SEQ ID NO: 3) 2E2 RKF F-Y mut (SEQ ID NO: 24)HCKFmut 2E2 RHC (SEQ ID NO: 4) 2E2 RKF F-Y mut (SEQ ID NO: 24) HDKFmut2E2 RHD (SEQ ID NO: 5) 2E2 RKF F-Y mut (SEQ ID NO: 24) HFKFmut 2E2 RHF(SEQ ID NO: 7) 2E2 RKF F-Y mut (SEQ ID NO: 24) HGKFmut 2E2 RHG (SEQ IDNO: 8) 2E2 RKF F-Y mut (SEQ ID NO: 24) RHE Y-VKA 2E2 RHE Y-V (SEQ ID NO:13) 2E2 RKA (SEQ ID NO: 16) RHE Y-VKB 2E2 RHE Y-V (SEQ ID NO: 13) 2E2RKB (SEQ ID NO: 17) RHE Y-VKC 2E2 RHE Y-V (SEQ ID NO: 13) 2E2 RKC (SEQID NO: 18) RHE Y-VKD 2E2 RHE Y-V (SEQ ID NO: 13) 2E2 RKD (SEQ ID NO: 19)RHE Y-VKE 2E2 RHE Y-V (SEQ ID NO: 13) 2E2 RKE (SEQ ID NO: 20) RHE Y-VKF2E2 RHE Y-V (SEQ ID NO: 13) 2E2 RKF (SEQ ID NO: 21) RHE Y-VKG 2E2 RHEY-V (SEQ ID NO: 13) 2E2 RKG (SEQ ID NO: 22) RHE E-DKA 2E2 RHE E-D (SEQID NO: 12) 2E2 RKA (SEQ ID NO: 16) RHE E-DKB 2E2 RHE E-D (SEQ ID NO: 12)2E2 RKB (SEQ ID NO: 17) RHE E-DKC 2E2 RHE E-D (SEQ ID NO: 12) 2E2 RKC(SEQ ID NO: 18) RHE E-DKD 2E2 RHE E-D (SEQ ID NO: 12) 2E2 RKD (SEQ IDNO: 19) RHE E-DKE 2E2 RHE E-D (SEQ ID NO: 12) 2E2 RKE (SEQ ID NO: 20)RHE E-DKF 2E2 RHE E-D (SEQ ID NO: 12) 2E2 RKF (SEQ ID NO: 21) RHE E-DKG2E2 RHE E-D (SEQ ID NO: 12) 2E2 RKG (SEQ ID NO: 22) RHE E-DKFmut 2E2 RHEE-D (SEQ ID NO: 12) 2E2 RKF F-Y mut (SEQ ID NO: 24) RHE S-GKA 2E2 RHES-G (SEQ ID NO: 11) 2E2 RKA (SEQ ID NO: 16) RHE S-GKB 2E2 RHE S-G (SEQID NO: 11) 2E2 RKB (SEQ ID NO: 17) RHE S-GKC 2E2 RHE S-G (SEQ ID NO: 11)2E2 RKC (SEQ ID NO: 18) RHE S-GKD 2E2 RHE S-G (SEQ ID NO: 11) 2E2 RKD(SEQ ID NO: 19) RHE S-GKE 2E2 RHE S-G (SEQ ID NO: 11) 2E2 RKE (SEQ IDNO: 20) RHE S-GKF 2E2 RHE S-G (SEQ ID NO: 11) 2E2 RKF (SEQ ID NO: 21)RHE S-GKG 2E2 RHE S-G (SEQ ID NO: 11) 2E2 RKG (SEQ ID NO: 22) RHETriple-KA 2E2 RHE triple (SEQ ID NO: 14) 2E2 RKA (SEQ ID NO: 16) RHETriple-KB 2E2 RHE triple (SEQ ID NO: 14) 2E2 RKB (SEQ ID NO: 17) RHETriple-KC 2E2 RHE triple (SEQ ID NO: 14) 2E2 RKC (SEQ ID NO: 18) RHETriple-KD 2E2 RHE triple (SEQ ID NO: 14) 2E2 RKD (SEQ ID NO: 19) RHETriple-KE 2E2 RHE triple (SEQ ID NO: 14) 2E2 RKE (SEQ ID NO: 20) RHETriple-KF 2E2 RHE triple (SEQ ID NO: 14) 2E2 RKF (SEQ ID NO: 21) RHETriple-KG 2E2 RHE triple (SEQ ID NO: 14) 2E2 RKG (SEQ ID NO: 22) RHETriple-KFmut 2E2 RHE triple (SEQ ID NO: 14) 2E2 RKF F-Y mut (SEQ ID NO:24) RHE Y-VKFmut 2E2 RHE Y-V (SEQ ID NO: 13) 2E2 RKF F-Y mut (SEQ ID NO:24) RHE E-DKFmut 2E2 RHE E-D (SEQ ID NO: 12) 2E2 RKF F-Y mut (SEQ ID NO:24)

TABLE 4 Amino acid sequences of HVRs from 2E2 andhumanized antibody variants Antibody Chain HVR1 HVR2 HVR3 2E2 antibodyHeavy IYGAH VIWAGGSTNYNSALMS DGSSPYYYSMEY chain SEQ ID NO: 61SEQ ID NO: 62 SEQ ID NO: 63 Light SATSSVSYMH STSNLAS QQRSSYPFT chainSEQ ID NO: 64 SEQ ID NO: 65 SEQ ID NO: 66Humanized Heavy Chain Variants 2E2 RHA, 2E2 RHB,2E2 RHC, 2E2 RHD, 2E2 RHE, 2E2 RHF, 2E2 RHG, 2E2 RHA2, and 2E2RHB2 HeavyIYGAH VIWAGGSTNYNSALMS DGSSPYYYSMEY chain SEQ ID NO: 61 SEQ ID NO: 62SEQ ID NO: 63 Humanized Light Chain Variants 2E2 RKA, 2E2 RKB,2E2 RKC, 2E2 RKD, 2E2 RKE, 2E2 RKF, and 2E2 RKG Light SATSSVSYMH STSNLASQQRSSYPFT chain SEQ ID NO: 64 SEQ ID NO: 65 SEQ ID NO: 66Humanized Heavy Chain Variants 2E2 RHE S-G,2E2 RHE E-D, 2E2 RHE Y-V, and 2E2 RHE triple 2E2 RHE IYGAHVIWAGGSTNYNSALMS DGSSPYYYGMEY S-G SEQ ID NO: 61 SEQ ID NO: 62SEQ ID NO: 67 2E2 RHE IYGAH VIWAGGSTNYNSALMS DGSSPYYYSMDY E-DSEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 68 2E2 RHE IYGAH VIWAGGSTNYNSALMSDGSSPYYYSMEV Y-V SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 69 2E2 RHE IYGAHVIWAGGSTNYNSALMS DGSSPYYYGMDV triple SEQ ID NO: 61 SEQ ID NO: 62SEQ ID NO: 70 Humanized Light Chain Variants 2E2 RKA F-Y and 2E2 RKF F-Y2E2 RKA SATSSVSYMH STSNLAS QQRSSYPYT F-Y SEQ ID NO: 64 SEQ ID NO: 65SEQ ID NO: 71 2E2 RKF SATSSVSYMH STSNLAS QQRSSYPYT F-Y SEQ ID NO: 64SEQ ID NO: 65 SEQ ID NO: 71

TABLE 5 Amino acid sequences of FRs from 2E2 andhumanized antibody variants FR1 FR2 FR3 FR4 Heavy Chain 2E2QVQLKESGPGLVA WVRQPPGKGLEW RLSISKDNSKSQVF WGQGTSVTVSS PSQSLSITCTVSGLG (SEQ ID LKINSLQTDDTALY (SEQ ID NO: FSLT (SEQ ID NO: 30) YCAR 44)NO: 25) (SEQ ID NO: 37) 2E2 RHA EVQLVESGGGLVQ WVRQAPGKGLEWRFTISKDNSKNTVY WGQGTTVTVSS PGGSLRLSCAASG VS (SEQ ID LQMNSLRAEDTAVY(SEQ ID NO: FSLT (SEQ ID NO: 31) YCAR 45) NO: 26) (SEQ ID NO: 38)2E2 RHB EVQLVESGGGLVQ WVRQAPGKGLEW RLSISKDNSKNTVY WGQGTTVTVSSPGGSLRLSCAVSG LG (SEQ ID LQMNSLRAEDTAVY (SEQ ID NO: FSLT (SEQ ID NO: 32)YCAR 45) NO: 27) (SEQ ID NO: 39) 2E2 RIK EVQLVESGGGLVQ WVRQAPGKGLEWRFTISKDNSKNTVY WGQGTTVTVSS PGGSLRLSCAVSG VS (SEQ ID LQMNSLRAEDTAVY(SEQ ID NO: FSLT (SEQ ID NO: 31) YCAR 45) NO: 27) (SEQ ID NO: 38)2E2 RHD EVQLVESGGGLVQ WVRQAPGKGLEW RFTISKDNSKNTVY WGQGTTVTVSSPGGSLRLSCAASG LS (SEQ ID LQMNSLRAEDTAVY (SEQ ID NO: FSLT (SEQ ID NO: 33)YCAR 45) NO: 26) (SEQ ID NO: 38) 2E2 RHE EVQLVESGGGLVQ WVRQAPGKGLEWRFTISKDNSKNTVY WGQGTTVTVSS PGGSLRLSCAASG VG (SEQ ID LQMNSLRAEDTAVY(SEQ ID NO: FSLT (SEQ ID NO: 34) YCAR 45) NO: 26) (SEQ ID NO: 38)2E2 RHF EVQLVESGGGLVQ WVRQAPGKGLEW RLTISKDNSKNTVY WGQGTTVTVSSPGGSLRLSCAASG VS (SEQ ID LQMNSLRAEDTAVY (SEQ ID NO: FSLT (SEQ ID NO: 31)YCAR 45) NO: 26) (SEQ ID NO: 40) 2E2 RHG EVQLVESGGGLVQ WVRQAPGKGLEWRFSISKDNSKNTVY WGQGTTVTVSS PGGSLRLSCAASG VS (SEQ ID LQMNSLRAEDTAVY(SEQ ID NO: FSLT (SEQ ID NO: 31) YCAR 45) NO: 26) (SEQ ID NO: 41)2E2 RHA2 QVQLQESGPGLVK WIRQPPGKGLEW RVTISVDTSKNQFS WGQGTLVTVSSPSETLSLTCTVSG IG (SEQ ID LKLSSVTAADTAVY (SEQ ID NO: GSIS (SEQ ID NO: 35)YCAR 46) NO: 28) (SEQ ID NO: 42) 2E2 RHB2 QVQLQESGPGLVK WVRQPPGKGLEWRLSISKDNSKNQVS WGQGTLVTVSS PSETLSLTCTVSG LG (SEQ ID LKLSSVTAADTAVY(SEQ ID NO: FSLT (SEQ ID NO: 36) YCAR 46) NO: 29) (SEQ ID NO: 43)2E2 RHE S-G EVQLVESGGGLVQ WVRQAPGKGLEW RFTISKDNSKNTVY WGQGTTVTVSSPGGSLRLSCAASG VG (SEQ ID LQMNSLRAEDTAVY (SEQ ID NO: FSLT (SEQ ID NO: 34)YCAR 45) NO: 26) (SEQ ID NO: 38) 2E2 RHE E-D EVQLVESGGGLVQ WVRQAPGKGLEWRFTISKDNSKNTVY WGQGTTVTVSS PGGSLRLSCAASG VG (SEQ ID LQMNSLRAEDTAVY(SEQ ID NO: FSLT (SEQ ID NO: 34) YCAR 45) NO: 26) (SEQ ID NO: 38)2E2 RHE Y-V EVQLVESGGGLVQ WVRQAPGKGLEW RFTISKDNSKNTVY WGQGTTVTVSSPGGSLRLSCAASG VG (SEQ ID LQMNSLRAEDTAVY (SEQ ID NO: FSLT (SEQ ID NO: 34)YCAR 45) NO: 26) (SEQ ID NO: 38) 2E2 RHE EVQLVESGGGLVQ WVRQAPGKGLEWRFTISKDNSKNTVY WGQGTTVTVSS triple PGGSLRLSCAASG VG (SEQ IDLQMNSLRAEDTAVY (SEQ ID NO: FSLT (SEQ ID NO: 34) YCAR 45) NO: 26)(SEQ ID NO: 38) Light Chain 2E2 QIILTQSPAIMSA WFQQKPGTSPKLGVPVRFSGSGSGTS FGSGTKLEIK SPGEKVSITC WIY (SEQ ID YSLTISRMEAEDAA(SEQ ID NO: (SEQ ID NO: NO: 50) TYYC 59) 47) (SEQ ID NO: 54) RKAEIVLTQSPATLSL WFQQKPGQAPRL GIPARFSGSGSGTD FGPGTKLDIK SPGERATLSCLIY (SEQ ID FTLTISSLEPEDFA (SEQ ID NO: (SEQ ID NO: NO: 51) VYYC 60) 48)(SEQ ID NO: 55) RKB EIILTQSPATLSL WFQQKPGQAPRL GVPARFSGSGSGTD FGPGTKLDIKSPGERATLSC WIY (SEQ ID YTLTISSLEPEDFA (SEQ ID NO: (SEQ ID NO: NO: 52)VYYC 60) 49) (SEQ ID NO: 56) RKC EIILTQSPATLSL WFQQKPGQAPRLGIPARFSGSGSGTD FGPGTKLDIK SPGERATLSC LIY (SEQ ID FTLTISSLEPEDFA(SEQ ID NO: (SEQ ID NO: NO: 51) VYYC 60) 49) (SEQ ID NO: 55) RKDEIVLTQSPATLSL WFQQKPGQAPRL GIPARFSGSGSGTD FGPGTKLDIK SPGERATLSCWIY (SEQ ID FTLTISSLEPEDFA (SEQ ID NO: (SEQ ID NO: NO: 52) VYYC 60) 48)(SEQ ID NO: 55) RKE EIVLTQSPATLSL WFQQKPGQAPRL GVPARFSGSGSGTD FGPGTKLDIKGSPERATLSC LIY (SEQ ID FTLTISSLEPEDFA (SEQ ID NO: (SEQ ID NO: NO: 51)VYYC 60) 48) (SEQ ID NO: 57) RKF EIVLTQSPATLSL WFQQKPGQAPRLGIPARFSGSGSGTD FGPGTKLDIK GSPERATLSC LIY (SEQ ID YTLTISSLEPEDFA(SEQ ID NO: (SEQ ID NO: NO: 51) VYYC 60) 48) (SEQ ID NO: 58) RKGEIVLTQSPATLSL WYQQKPGQAPRL GIPARFSGSGSGTD FGPGTKLDIK SPGERATLSCLIY (SEQ ID FTLTISSLEPEDFA (SEQ ID NO: (SEQ ID NO: NO: 53) VYYC 60) 48)(SEQ ID NO: 55) RKAF-Y EIVLTQSPATLSL WFQQKPGQAPRL GIPARFSGSGSGTDFGPGTKLDIK GSPERATLSC LIY (SEQ ID FTLTISSLEPEDFA (SEQ ID NO: (SEQ ID NO:NO: 51) VYYC 60) 48) (SEQ ID NO: 55) RKFF-Y EIVLTQSPATLSL WFQQKPGQAPRLGIPARFSGSGSGTD FGPGTKLDIK SPGERATLSC LIY (SEQ ID YILTISSLEPEDFA(SEQ ID NO: (SEQ ID NO: NO: 51) VYYC 60) 48) (SEQ ID NO: 55)

Clones were sequenced and expression plasmid DNA was prepared using thePlasmid Miniprep Kit (Qiagen) or PureYield Plasmid Maxiprep System(Promega) according to the manufacturer's protocol. Expression plasmidpreparations encoding humanized or chimeric VH and VK were used totransfect Expi293 cells (Invitrogen) as described above in Example 1.The cells were cultured for 7 days in serum free media, whereupon theconditioned medium from the cells was harvested and assayed by ELISA toconfirm production of the antibodies.

Siglec-8 Binding By Antibodies Encoded By Humanized VH and VK Constructs

Basic humanized heavy and light chains were paired with their chimericcounterparts in an attempt to identify any gross problems with thehumanization design. Siglec-8 antigen was used to measure antibodybinding by ELISA as described in Example 1. 2E2RHB, when paired with thechimeric light chain, appeared to be more potent than the RHA heavychain, while RKB construct in association with the heavy chain chimericconstruct (cVH) bound Siglec-8 antigen with a higher potency then boththe paired cVK constructs. These results confirmed that humanizationdesign for both heavy and light chains was approximately correct and allbound Siglec-8 but that further work was required to identify additionalhumanized antibodies with better binding characteristics.

Siglec-8 Binding By Fully Humanized Antibodies Combined with ChimericCounterpart

The fully humanized antibody heavy and light chains were combined andcompared with the chimeric antibody to determine if replacing the 4 Åand canonical framework residues and interface residues was sufficientto successfully humanize 2E2. Expi293 cells were co-transfected withcombinations of different humanized light chain vectors in associationwith different humanized heavy chain vectors. Siglec-8 antigen was usedto measure antibody binding by ELISA as described in Example 1. Thebinding of these antibodies to Siglec8 as compared to the chimericcontrol showed that HBKA and HBKB appeared to be better combinationsthan HAKA and HAKB (Table 6).

Siglec-8 Binding By Fully Humanized Antibodies

In order to determine the relative importance of the single amino acidsubstitutions in the heavy chain, the humanized heavy chains RHC (A24V),RHD (V48L), RHE (S49G), RHF (F67L) and RHG (T68S) were expressed incombination with all light chain variants and compared to RHA and RHBversions and the chimeric antibody control. The results of theseantibodies binding to Siglec-8 ECD suggested that all these humanizedantibody combinations bound Siglec-8 at the same potency except forfamilies RHA, RHF and RHG (with all light chain variants) (Table 6).

It was next determined if changing the light chain from RKA to RKB, withsingle amino acid substitutions, would affect antibody binding. Bindingof antibodies consisting of combinations of all heavy chain variantswith light chains RKC (V3I), RKD (V48L), RKE (L47W) and RKF (E58V),compared to the chimeric antibody and the RKA and RKB versions wasassessed. There did not appear to be a light chain variant thatsignificantly affected the pattern of binding (Table 6). In addition,the relevance of the light chain germline residue F71Y (RKG) was alsoexamined in combination with all heavy chain variants and the resultsdemonstrated that this residue generally caused a great decline inbinding.

It appeared that HEKA and HEKF were the best candidate antibodiesagainst Siglec-8. Therefore an ELISA was performed to re-test theantibodies that bound with the highest potency to the antigen comparedto both the chimeric antibody and HEKA and HEKF as controls. The resultsindicated that the different combinations of humanized heavy andhumanized light chains bound in a similar way to Siglec-8, apart fromcombinations with RHA (Table 6). The results highlighted that HEKA andHEKF were very good candidates and compared favorably with the chimericpositive control and had minimal mouse residues in the frameworks.

Generation of 2E2RH Version 2 and CDR Mutated Variants

A subsequent humanized heavy chain was generated based on the closestgermline gene. The IGHV4-59 germline sequence, the most similar germlineto 2E2VH (FIG. 1), was used to design the graft of the mouse CDRs,synthesized (GenScript) and prepared in the same manner as the otherconstructs described above, to generate humanized antibodies forcomparison with the first version of RHA and RHB chains. In addition, todetermine if the antibody could tolerate certain CDR3 residues beingchanged to germline. Three mutations were introduced in the CDR3 of theRHE variant heavy chain (single and triple mutations) and one mutationin the CDR3 of RKA and RKF light chain (FIG. 3) by site-directedmutagenesis. Antibodies containing RHE mutated or RKA/RKF mutated wereexpressed and combined with the complete panel of the complementarychain using the same method described above.

The heavy chain CDRs grafted into human germline was compared with theother heavy chain versions. The straight graft (RHA2) completelydisrupted binding to the antigen and the germline framework with the 4Å/VCI residues back mutated to mouse (RHB2) behaved very similarly tothe first RHB variant but contained 10 mouse residues as compared to the5 of RHB. The binding data illustrated the influence of introducingmutations in CDR3 of both chains and suggested that the mutations in theheavy chain had a detrimental effect on binding to Siglec-8 while thelight chain mutation on its own also did not provide much improvement,although the best antibodies were the ones containing RHB (all mouseback mutations) and RHE, the heavy chain candidate (Table 6).

TABLE 6 Antibody binding to human Siglec-8 ECD. EC50 EC50 EC50 EC50Antibody (nM) Antibody (nM) Antibody (nM) Antibody (nM) HumanizedAntibodies HAKA 0.058 HCKA 0.056 HEKA 0.112 HGKA 0.061 HAKB 0.071 HCKB0.065 HEKB 0.057 HGKB 0.134 HAKC 0.066 HCKC 0.058 HEKC 0.065 HGKC 0.065HAKD 0.058 HCKD 0.049 HEKD 0.070 HGKD 0.047 HAKE 0.052 HCKE 0.059 HEKE0.205 HGKE 0.116 HAKF 0.062 HCKF 0.050 HEKF 0.097 HGKF 0.088 HAKG 0.189HCKG 0.273 HEKG 0.302 HGKG 0.254 HBKA 0.070 HDKA 0.067 ch2E2 0.062 VHVK0.043 Purif. HBKB 0.056 HDKB 0.055 cVHKA NA HBKC 0.033 HDKC 0.057 cVHKB0.027 HBKD 0.039 HDKD 0.048 HAcVK 0.067 HBKE 0.061 HDKE 0.065 HBcVK0.066 HBKF 0.051 HDKF 0.057 ch2E2 0.062 Purif. HBKG 0.212 HDKG 0.113ch2E2 0.044 ch2E2 0.062 Purif. Purif. ch2C4 0.057 ch2C4 0.079 ch2C4 T10.075 HFKA 0.106 HAKA 0.085 HA2KA NA HFKB 0.099 HAKF 0.078 HA2KB 124.30HFKC 0.063 HBKA 0.055 HB2KA NA HFKD 0.058 HBKF 0.066 HB2KB 0.051 HFKE0.104 HA2KF 8.719 ch2C4 0.069 HFKF 0.118 HB2KF 0.063 HA2KC 0.488 HFKG0.355 HEKA 0.068 HA2KD NA HEKB 0.063 HA2KE NA HEKC 0.069 HA2KF NA HEKD0.059 HA2KG NA HEKE 0.057 HB2KC 0.101 HEKF 0.064 HB2KD 0.075 HEKF T20.064 HB2KE 0.087 mo2C4 0.115 HB2KF 0.089 HB2KG 0.227 CDR Human AntibodyVariants ch2C4 0.052 ch2C4 0.208 ch2C4 0.057 RHE Y-V 0.088 RHE 50.620HAKFmut 0.086 KA S-GKA RHE Y-V 0.306 RHE 626.900 HBKFmut 0.048 KB S-GKBRHE Y-V 0.215 RHE 173.300 HCKFmut 0.078 KC S-GKC RHE Y-V 0.283 RHE 8.647HDKFmut 0.056 KD S-GKD RHE Y-V 0.070 RHE NA HEKFmut 0.072 KE S-GKE RHEY-V 0.091 RHE 3.279 HFKFmut 0.057 KF S-GKF RHE Y-V 8.808 RHE 33.540HFKFmut 0.062 KG S-GKG RHE E-D 0.388 RHE 18.640 KA Triple-KA RHE E-D0.289 RHE NA KB Triple-KB RHE E-D 0.516 RHE NA KC Triple-KC RHE E-D0.316 RHE 13.200 KD Triple-KD RHE E-D 0.364 RHE 34.060 KE Triple-KE RHEE-D 0.445 RHE NA KE Triple-KF RHE E-D NA RHE NA KG Triple-KG ch2C4 0.075ch2C4 0.075 ch2C4 0.175 HEKA 0.054 HA2KA NA RHE S-G 207.100 KFmut HEKE0.042 HA2KB 124.300 RHE Triple NA KFmut HEICF 0.074 HA2KFmut 31.610HEICF 0.052 RHE Y-V 0.398 HB2KA 0.051 HEKA 0.064 KFmut RHE E-D 1.399HB2KB 0.069 KFmut HB2KFmut 0.080 Note: Each column represents oneexperiment; NA indicates not available.

Thermo Stability of Humanized Candidate Antibodies to High Temperatures

The thermo stability of the humanized antibodies was compared. Theantibodies were subjected to higher temperatures, varying from −20 to85° C. for 10 minutes, cooled to room temperature and assessed in anELISA assay at an EC80 concentration of each candidate. The leadantibody candidates appeared stable (FIG. 4). The HEKA/KF with CDRL3(i.e., CDR3 of the light chain) mutated antibodies were completelyinactive at 68° C., while the chimeric was inactive at 70° C., at whichtemperature the lead candidates HEKA and HEKF were still 25-50% active,only showing complete inactivity at 75° C.

Determination of Humanized Candidate Antibodies Tm

In order to determine the melting temperature of the lead antibodies,the chimeric, HEKA, HEKF and the same humanized candidates with theCDRL3 (i.e., CDR3 of the light chain) mutation were purified in a 2-stepaffinity chromatography and gel filtration system and tested in athermal shift assay. Antibodies were incubated at two differentconcentrations with a fluorescent dye (Sypro Orange) for 71 cycles with1° C. increase per cycle in a qPCR thermal cycler. Tm was defined astemperature for 50% maximal fluorescence. Tm for the chimeric and thefive humanized antibodies confirmed the results obtained in the thermostability assay: the most stable antibodies were HEKA and HEKF having ahigher Tm than the other humanized antibodies tested. HEKA has a higherTm than the chimeric antibody (FIG. 5 and Table 7).

TABLE 7 Tm of chimeric and humanized antibodies Antibody TM chVHVK 2 uM71° C. chVHVK 1 uM 71° C. HEKA 2 uM 72° C. HEKA 1 uM 72° C. HEKF 2 uM70° C. HEKF 1 uM 70° C. HEKAmut 2 uM 68° C. HEKAmut 1 uM 68° C. HEKFmut2 uM 67° C. HEKFmut 1 uM 68° C.

Affinity and Avidity of Humanized Candidate Antibodies

Antibody avidity determination was carried out by SPR analysis using aBiacore T200. Binding of human Siglec-8 ECD protein to mouse, chimericand humanized anti-Siglec-8 antibodies was measured on a Biacore T100.The capture antibodies (goat-anti-human-Fc and goat-anti-mouse-Fc fromJackson Immunoresearch) were immobilized on a CM5 chip according tomanufacturer's protocol (Biacore, GE). Flow-cell 1, 2 and 3 wereimmobilized with anti-human, and flow cell 4 with anti-mouse antibodies.The assay was conducted at 25° C., at a flow rate of 30 μl/min. Assaybuffer was 20 mM Tris-HCl pH 8.3, 150 mM Sodium Chloride, 0.05%Polysorbate 20, 10% glycerol, 0.1% BSA, made in ultrapure water. DimericSiglec-8 (impurities of monomeric and oligomeric Siglec-8 were removedby size-exclusion chromatography) was diluted in assay buffer from 15 nMto 1.88 pM with 2× dilutions. Antibodies were captured to a change ofapproximately 120 RU. Six-minute high performance injections wereconducted, followed by 120-minute dissociations. Flow cells wereregenerated with 50 mM glycine pH 1.5. Results were double blanked withan empty reference cell and multiple assay buffer injections, andanalyzed with 1:1 global fit parameters.

The avidity of murine 2E2 and chimeric 2E2 antibodies was determined tobe 28 pM and 16 pM, respectively (Table 8). The avidities for thehumanized antibodies were 17 pM for HEKA and 21 pM for HEKF whichindicated that the humanization had successfully retained and enhancedthe binding activity.

TABLE 8 Avidity determination of mouse, chimeric and humanizedantibodies Antibody ka (1/Ms) kd (1/s) KD (pM) mouse 2E2 5.56E+051.54E−05 28 chimeric 2E2 8.51E+05 1.32E−05 16 HEKA 6.38E+05 1.11E−05 17HEKF 6.78E+05 1.40E−05 21

Antibody affinity determination was also carried out by bio-layerinterferometry (ForteBio). Binding of mouse, chimeric and humanizedanti-Siglec-8 antibody Fab fragments to human Siglec-8 protein wasmeasured on a ForteBio Octet Red 384. The assay was conducted at 25° C.,at an RPM of 1000 in assay buffer. HBS buffer with 1× ForteBio Kineticsbuffer was made from stock solutions (Biacore BR-10670, ForteBio18-132respectively) in ultrapure water. Fab fragments (antibodies weredigested with Thermo-Pierce immobilized papain per manufacturer'sspecifications) were diluted in assay buffer from 50 nM to 1.56 nM with2× dilutions. The Siglec-8-Fc tagged protein was immobilized onAnti-Human-Capture sensors at 100 nM in assay buffer for 3 min to achange in nm of approximately 1.2. Two-minute associations wereconducted, followed by 10-minute dissociations. Results were blankedwith an empty reference AHC sensor, and analyzed in ForteBio analysissoftware with 1:1 global fit parameters.

The affinity of murine 2E2 and chimeric 2E2 Fab fragments was determinedto be 536 pM and 585 pM, respectively (Table 9). The affinities for thehumanized antibodies were 464 pM for HEKA and 592 pM for HEKF whichindicated that the humanization had successfully retained the bindingactivity for these two humanized antibodies. HEKA had a highermonovalent affinity for Siglec-8 than mouse and chimeric 2E2 in thisassay. The affinities for the humanized antibody variants, HEKAmut andHEKFmut, also where in the picomolar range with a KD of 902 pM and 1160pM, respectively.

TABLE 9 Affinity determination of mouse, chimeric and humanizedantibodies Antibody kon (1/Ms) kdis (1/s) KD (pM) mouse 2E2 1.14E+066.12E−04 536 chimeric 2E2 9.51E+05 5.56E−04 585 HEKA 1.04E+06 4.82E−04464 HEKF 9.20E+05 5.45E−04 592 HEKAmut 7.26E+05 6.55E−04 902 HEKFmut4.45E+05 5.16E−04 1160 

Solubility of Humanized Candidate Antibodies

The purified chimeric and candidate antibodies were sequentiallyconcentrated using centrifugal filter devices (Amicon 30K 4 mL, 4000 g 5min—first concentration; Amicon Ultra 0.5 mL 3K, 14000 g—followingconcentrations) and the concentration measured at each step. All of thesamples were concentrated in total by a factor of up to 21-24 withoutprecipitating and tested by ELISA which showed that none had lostbinding potency to Siglec-8. The antibodies were not prone toprecipitation at concentrations up to at least 25 mg/mL. Specifically,solubility for ch2E2 was at least 18 mg/mL, for HEKA was at least 25mg/mL, for HEKF was at least 8 mg/mL, for HEKAmut at least 29 mg/mL, andHEKFmut at least 17 mg/mL.

Aggregation of Humanized Candidate Antibodies

Samples were filtered prior to analysis to remove any salt or proteinprecipitation and concentrations were re-measured. They were theninjected at 0.4 mL/min into a size exclusion column in an HPLC systemand analyzed by multi-angle light scattering to determine the absolutemolar masses and checked for aggregation. All variants showed no signsof aggregation with an average molecular weight ranging from 134.9-138.2kDa, which was the expected range for an IgG monomer in this analysis(Table 10).

All samples were monodispersed (Mw/Mn<1.05). However, the distributionanalysis plots showed the presence of glycosylation variants (ch2C4,HEKA and HEKFMut). The distribution analysis plots also show thepresence of a ˜105-120 kDa species in all the samples, which could havebeen disintegrated antibody or a low glycosylation variant. The massrecoveries were between 82.9-102.8% (calculated mass over injectedmass), which indicated good protein recovery and the samples did notseem to stick to the column or contain insoluble aggregates, which wouldbe retained by the guard column. Overall the data indicated there was nosignificant aggregation in any of the anti-Siglec-8 antibody samplesanalyzed (Table 10).

TABLE 10 Aggregation analysis of chimeric and humanized variantantibodies Poly- Cal- Mass dis- cu- Mass re- persity lated frac- cov- MWUn- (Mw/ Un- mass tion ery (kDa) certainty Mn) certainty (μg) (%) (%)ch2C4 138.7 0.80% 1.01 1.10% 11.02 100 96.6 ch2C4 134.6 0.80% 1.0171.10% 10.86 100 95.2 Average 136.6 1.013 10.94 100 95.9 % standard 2.10.445 0.11 0 1 deviation HEKA 134 0.90% 1.03 1.30% 15 100 102.8 HEKA135.9 0.90% 1.01 1.30% 14.4 100 98.6 Average 134.9 1.02 14.7 100 100.7 %standard 1 0.014 0.43 0 2.9 deviation HEKA Mut 135.4 0.80% 1.013 1.10%13.61 100 98.8 HEKA Mut 137.3 0.90% 1.011 1.30% 13.68 100 99.3 Average136.3 1.012 13.65 100 99 % standard 1.3 0.001 0.05 0 0.4 deviation HEKF291 13.90%  1.742 14.40%  4.83 100 88.1 HEKF 138.2 0.80% 1.01 1.10% 4.54100 82.9 Average 214.6 1.376 4.69 100 85.5 % standard 50.3 37.622 4.31 04.3 deviation

Freeze-Thaw Stability of Humanized Candidate Antibodies

Purified chimeric ch2C4 antibody, humanized HEKA and HEKF antibodies,and humanized HEKAmut and HEKFmut antibody variants were subjected to−20° C. for 60 minutes, thawed at room temperature and used in an ELISAassay at the EC80 concentration for each candidate. HEKA showed thehighest stability in this assay (FIG. 6).

ADCC Activity of Non-Fucosylated Antibodies

Materials

RBC Lysis Buffer (10× RBC Lysis Buffer): Dilute to 1× as directed bymanufacturer (eBioscience, 00-4300-54).

PBS: DPBS without Ca²⁺/Mg²⁺ (Hyclone, SH30028.02).

Complete RPMI: Sterile Filtered RPMI-1640 (Invitrogen) with 10% FBS.

96-well U-Bottom Plate (Falcon, 353077).

LDH Assay: CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega,G1780)

FIX Buffer: 1-4% paraformaldehyde in PBS. Prepare from 16%paraformaldehyde (EM grade, methanol-free) by diluting in PBS (ElectronMicroscopy Diatom, 50-980-488).

Methods

To test ADCC and apoptotic activity of anti-Siglec-8 antibodies oneosinophils, fresh peripheral blood leukocytes (PBLs) were incubatedwith chimeric and mouse 2E2 antibodies. Low-fucose chimeric 2E2 IgG1antibody showed the most potent killing of eosinophils and hadsignificantly higher potency than fucosylated chimeric 2E2 IgG1consistent with higher ADCC activity for the low-fucose form of theantibody (FIG. 7).

For evaluation of anti-Siglec-8 antibody activity in total peripheralblood leukocytes, PBLs are obtained by standard methods from donor bloodcollected less than 24 hours since harvest and are resuspended inComplete RPMI medium. Cells are counted and adjusted to 10×10⁶/mL inComplete RPMI medium and plated at 100 μL/well in a sterile 96-wellU-bottom plate. Anti-Siglec-8 antibody is added at a concentrationbetween 0.0001 ng/mL to 10 μg/mL. Plates are centrifuged at 200 g for 1minute and incubated in a humidified 37° C. incubator at 5% CO₂ for >4hours. The cell populations are evaluated by flow cytometry to assessdepletion of eosinophils and basophils for example using reagents shownin Table 11 and evaluated by flow cytometry. Removal of CCR3-positive,CD16-negative granular cells (high side-scatter) cells can be used todetect depletion of eosinophils. Basophil counts can be determined forexample by analysis of CCR3-positive low side-scatter cells.

TABLE 11 Reagents Target Catalog Target Format Clone Species HostIsotype Vendor Number 7AAD 7AAD N/A N/A N/A N/A BD 559925 Annexin PE N/AN/A N/A N/A BD 559763 V CD117 APC A3C6E2 Human Mouse IgG₁ Miltenyi130-091-733 CD16 FITC 3G8 Human Mouse IgG₁ BD 555406 CD193 Alexa 5E8Human Mouse IgG_(2b) BD 558208 CCR3 Fluor ® 647 CDw125 PE A14 Human Mouse IgG₁ BD 555902 (IL-5Rα) Cleaved Alexa F21-852 Human Mouse IgG₁ BD558710 PARP Fluor ® 647 FCϵR1α FITC AER-37 Human Mouse IgG_(2b) Miltenyi130-095-978 CRA1 Isotype PE MOPC- N/A Mouse IgG₁ BD 555749 IgG₁ 21Isotype FITC 27-35 N/A Mouse IgG_(2b) BD 555742 IgG_(2b) Isotype APCeBMG2b N/A Mouse IgG_(2b) eBioscience 17- IgG_(2b) 4732

For the evaluation of the ability of anti-Siglec-8 antibodies to induceapoptosis in purified eosinophils, fresh buffy coat collected less than24 hours since harvest from a blood sample or an equivalent bloodproduct is used. Purification of eosinophils is conducted following themanufacturer's instructions (Miltenyi Eosinophil Isolation Kit,130-092-010). Purified eosinophils are resupended at 1×10⁶/mL inComplete RPMI medium and cultured in the presence or absence of IL-5 (ata concentration of about 1 ng/mL to about 50 ng/mL) overnight. Thefollowing day, cultured eosinophils are harvested by repeated washing ofthe plate or flask. The cells are centrifuged at 200-400 g for less than10 minutes and resuspended at 1×10⁶/mL in Complete RPMI medium.Eosinophils are plated at 100 μL/well in a sterile 96-well U-bottomplate. 100 uL of 2× reagents prepared in Complete RPMI medium are addedto each well and dilutions are prepared as described above. The platesare centrifuged at 200 g for 1 minute and incubated in a humidified 37°C. incubator at 5% CO₂ for ≥4 hours. Annexin-V staining is performedaccording to manufacturer instructions, and apoptotic and necrotic cellsare analyzed by flow cytometry.

For evaluation of ADCC and apoptotic activity of anti-Siglec-8antibodies on isolated mast cells, human mast cells are isolated fromhuman tissues according to published protocols (Guhl et al., Biosci.Biotechnol. Biochem., 2011, 75:382-384; Kulka et al., In CurrentProtocols in Immunology, 2001, (John Wiley & Sons, Inc.)) ordifferentiated from human hematopoietic stem cells, for example asdescribed by Yokoi et al., J Allergy Clin Immunol., 2008, 121:499-505.Purified mast cells are resuspended at 1×10⁶/mL in Complete RPMI mediumin a sterile 96-well U-bottom plate and incubated in the presence orabsence of anti-Siglec-8 antibodies for 30 minutes at concentrationsranging between 0.0001 ng/ml and 10 μg/ml. Samples are incubated for afurther 4 to 16 hours with and without purified natural killer (NK)cells or fresh PBL to induce ADCC. Cell-killing by apoptosis or ADCC isanalyzed by flow cytometry using fluorescent conjugated antibodies todetect mast cells (CD117 and FcεR1) and Annexin-V and 7AAD todiscriminate live and dead or dying cells. Annexin-V and 7AAD stainingare performed according to manufacturer's instructions.

Evaluation of Eosinophil-Depleting Activity of Humanized Antibodies InVitro

Humanized antibodies were evaluated for their ability to induce Siglec-8mediated depletion of eosinophils from normal human blood in vitro incomparison with the murine 2E2 antibody.

Peripheral blood leukocytes (PBL) from human donor blood collected lessthan 24 hours after harvest were resuspended in complete RPMI medium[(RPMI-1640 medium (Invitrogen, Catalog Number A10491-01) supplementedwith 10% Fetal Bovine Serum)]. Cells were adjusted to 10⁷ per mL inComplete RPMI medium supplemented with 50 ng/ml recombinant human IL-5(R&D Systems, Catalog Number 205-IL-025) and plated at 100 μL/well in asterile 96-well U-bottom plate. Anti-Siglec-8 antibodies were added atconcentrations ranging from 0.1 pg/mL and 10 μg/mL (i.e., 1 pg/mL, 10pg/mL, 0.1 ng/mL, 1 ng/mL, 10 ng/mL, 0.1 μg/mL, 1 μg/mL, and 10 μg/mL)to determine the dose response and concentration providing 50% maximaleosinophil depletion (EC₅₀). Plates were centrifuged at 200 g for 1minute and incubated in a humidified 37° C. incubator at 5% CO₂ for 16hours. Incubation of PBLs with anti-Siglec-8 antibodies for 16 hours inthe presence of IL-5 sensitized eosinophils to Siglec-8-mediatedapoptosis. The cell populations were evaluated by flow cytometry toassess depletion of eosinophils. Eosinophil depletion was detected bythe removal of CCR3-positive granular (high side-scatter) cells.

Each of the tested humanized antibodies, with the exception of theHEKAmut IgG1 antibody, showed equivalent or increased potency (i.e.,lower EC₅₀) compared with the mouse 2E2 antibody for depletion of humaneosinophils (Table 12). The antibody potency for depletion of humaneosinophils was not dependent on isotype as the HEKA IgG1 antibody andHEKA IgG4 antibody showed similar potency.

TABLE 12 Potency of humanized anti-Siglec-8 antibodies for depletion ofeosinophils in vitro Mean EC₅₀ (ng/ml) Antibody Isotype for eosinophildepletion 2E2 IgG1 6.8 HEKA IgG1 4.3 HEKA IgG4 3.9 HEKAmut IgG1 43.3HEKF IgG1 6.7 HEKFmut IgG1 6.9 Mean EC50 indicates mean half-maximalantibody concentrations required for eosinophil depletion from 2independent assays.Anti-Siglec-8 Antibodies with an Active Isotype Are Capable of InducingADCC-Mediated Killing of Human Mast Cells In Vitro and In Vivo.

To generate a humanized anti-Siglec-8 antibody with potent Fc-receptormediated ADCC activity, the HEKA IgG1 antibody was expressed from a CHOcell line deficient in fucosyl transferase-8 (Lonza, Potelligent CHOK1SVcells) to generate an antibody with carbohydrates lacking α1,6 fucose(i.e., non-fucosylated antibody). A murine monoclonal anti-Siglec-8antibody with a murine IgG2a isotype (i.e., 1C3 antibody) thatrecognizes a different extracellular region of Siglec-8 than the HEKAIgG1 antibody was generated as described in Example 3. The chimeric 1H10antibody contains the V-regions of mouse monoclonal antibody 1H10 andhuman IgG1 kappa constant regions. Chimeric 1H10 antibody was expressedfrom the human 293TS cell line cultured in the presence of 10 μMKifunensine to generate low fucose antibody and purified by Protein Aaffinity chromatography.

The ability of non-fucosylated HEKA IgG1 and chimeric 1H10 low-fucoseantibody to induce NK-cell-mediated ADCC activity against human mastcells was evaluated in vitro. Primary human mast cells were isolated bylavage of the peritoneal cavity of immunodeficient NSGS mice engraftedwith human hematopoietic stem cells. Mast cells were incubated for 48hours with 10 μg/ml of non-fucosylated HEKA IgG1 antibody, chimeric 1H10low-fucose antibody, HEKA IgG4 antibody or isotype control human IgG1antibody, and with purified human CD56⁺CD16⁺ NK effector cells at aneffector to target cell (E:T) ratio of 10.75:1. ADCC activity wasdetermined by LDH release using a CytoTox 96 Cytotoxicity Assay kit(Promega, Catalog Number G1780). Non-fucosylated HEKA IgG1 antibody andchimeric 1H10 low-fucose antibody induced marked ADCC-mediated killingof human mast cells after 48 hours (FIG. 8A). LDH release induced bynon-fucosylated or low fucose anti-Siglec-8 antibody was 38-53% of themaximum LDH release induced using lysis solution (Promega, CatalogNumber G1821).

The ability of anti-Siglec-8 antibodies to deplete Siglec-8-positivemast cells in vivo was evaluated in a transgenic mouse model in whichhuman Siglec-8 is selectively expressed on the surface of mast cells,eosinophils and basophils. Mice were treated twice by intra-peritonealinjection with 100 μg of HEKA IgG4 antibody, HEKA IgG1 non-fucosylatedhumanized antibody, murine 1C3 antibody (murine IgG2a isotype) or humanIgG1 isotype control antibody. The two intra-peritoneal injections wereadministered 48 hours apart and peritoneal mast cells were isolated bylavage of the peritoneum 48 hours after the second injection.Non-fucosylated HEKA IgG1 antibody and murine 1C3 antibodyadministration led to significant depletion of peritoneal mast cells. Incontrast, HEKA IgG4 antibody did not show significant mast celldepletion, indicating that an active isotype is required for in vivodepletion of mast cells (FIG. 8B). These results demonstrate that twodifferent anti-Siglec-8 antibodies with an active isotype, murine IgG2aisotype or human IgG1 non-fucosylated isotype, directed againstdifferent regions of the extracellular domain of Siglec-8, can depleteSiglec-8-positive mast cells in vivo.

These results were unexpected since Siglec-8 has been described to berapidly internalized and therefore not suitable for inducing ADCCactivity. See O'Reilly et al., Trends Pharmacol Sci., 2009,30(5):240-248.

Humanized Anti-Siglec-8 Inhibits an IgE-Induced Passive CutaneousAnaphylaxis Reaction Mediated By Human Mast Cells In Vivo

Immunodeficient mice capable of generating abundant human mast cellsafter engraftment with human hematopoietic stem cells (HSC) have beendescribed (Tanaka et al., J Immunol., 2012, 188(12):6145-55). The mousestrain designated NSGS (The Jackson Laboratory) is a derivative of thenonobese diabetic/severe combined immunodeficiency (NOD SCID) mouse witha deletion of the IL-2 receptor gamma-chain gene (NSG mouse). NSGS miceare additionally transgenic for 3 human cytokines (stem cell factor[SCF], IL-3, and GM-CSF) to facilitate engraftment with humanhematopoietic stem cells. Upon engraftment of NSGS mice, human CD34⁺cells generate human eosinophils and enhanced numbers of human mastcells. Both cell types in engrafted NSGS mice express Siglec-8 at levelscomparable to the levels on the corresponding cell types isolated fromhuman peripheral blood and tissues. Thus, these mice provide anattractive model for evaluation of activity of anti-Siglec-8 antibodieson human cells in vivo.

In order to evaluate the effect of anti-Siglec-8 antibodies on mast cellactivity in vivo, an IgE-mediated ear swelling model was established inhumanized NSGS mice. In this model, passive cutaneous anaphylaxis (PCA),a Type I hypersensitivity reaction, was induced by injection of specificmonoclonal anti-hapten IgE (anti-NP IgE) into one ear 24 hours prior tosystemic injection of hapten-conjugated bovine serum albumin (NP-BSA).Chimeric anti-NP IgE with a human epsilon constant region was used toensure human mast-cell specific responses to hapten were generated, andthe immediate and late-phase edematous responses were measured bychanges in ear thickness.

NSGS mice were engrafted with human CD34⁺ HSC eight to twelve weeksprior to the assay. Chimeric monoclonal anti-NP IgE with a humanconstant region was intradermally injected in a mouse at a dose of 100ng into the right ear to sensitize human but not mouse skin mast cellsand PBS was intradermally injected into the left ear. Twenty four hourslater, PCA was induced by intravenous injection of 0.5 mg NP-BSA. Themice were dosed by intravenous injection with 0.1 mg anti-Siglec-8antibody (i.e., HEKA IgG4 antibody) or human IgG4 isotype controlantibody 24 hours pre-sensitization or 2 hours post-sensitization withanti-NP IgE. Ear thickness was measured at time points up to four hourspost-induction and at 24 hours post-induction to determine theearly-phase and late-phase ear swelling response, respectively.

HEKA IgG4 antibody prevented or inhibited both early and late-phasecutaneous allergic reactions in this PCA in vivo model (FIG. 9). In thismodel, the early-phase reaction is dependent on mast-cell degranulationand histamine release while the late-phase reaction is dependent on mastcell secretion of de novo synthesized mediators, including cytokines, aswell as eosinophil and basophil infiltration. HEKA IgG4 antibody alsoprevented or inhibited the PCA response in humanized NSGS mice whendosed either 24 hours pre-sensitization or 2 hours post-sensitizationwith anti-NP IgE (FIG. 9). No adverse effects of antibody treatment wereobserved during the course of these experiments.

Example 3: Generation and Characterization of Murine Anti-Siglec-8Antibodies

The extracellular region of Siglec-8 is composed of threeimmunoglobulin-like domains: a unique N-terminal V-set domain (Domain 1)that binds ligands, followed by two C-set domains (Domains 2 and 3).Antibody 1C3 is a murine monoclonal antibody with an IgG2a heavy chainand kappa light chain raised against a recombinant extracellular domainof human Siglec-8 (SEQ ID NO:74). Monoclonal antibodies 1H10 and 4F11are murine IgG1 heavy chain and kappa light chain antibodies raisedagainst a recombinant extracellular domain of human Siglec-8 (SEQ IDNO:74). See Table 13. These antibodies were identified from a hybridomascreen for antibodies that bound to recombinant Siglec-8 sequences fromhuman (SEQ ID NO:74) and non-human primates (SEQ ID NO:118).

TABLE 13 Amino acid sequences of HVRs from murine 1C3, 1H10,and 4F11 antibodies Anti- body Chain HVR1 HVR2 HVR3 1C3 Heavy SYAMSIISSGGSYT HETAQAAWFAY Chain SEQ ID YYSDSVKG SEQ ID NO: 94 NO: 88 SEQ IDNO: 91 1H10 Heavy DYYMY RIAPEDGDT EGNYYGSSILDY Chain SEQ ID EYAPKFQGSEQ ID NO: 95 NO: 89 SEQ ID NO: 92 4F11 Heavy SSWMN QIYPGDDYT LGPYGPFADChain SEQ ID NYNGKFKG SEQ ID NO: 96 NO: 90 SEQ ID NO: 93 1C3 LightSASSSV DTSKLAY QQWSSNPPT Chain SYMH SEQ ID SEQ ID NO: 103 SEQ ID NO: 100NO: 97 1H10 Light RASQDI FTSRLHS QQGNTLPWT Chain TNYLN SEQ IDSEQ ID NO: 104 SEQ ID NO: 101 NO: 98 4F11 Light SASSSV DTSSLAS QQWNSDPYTChain SYMY SEQ ID SEQ ID NO: 105 SEQ ID NO: 102 NO: 99

To identify the region comprising the epitope for anti-Siglec-8antibodies, fusion proteins containing each of the Siglec-8extracellular domains fused to human Ig-Fc were expressed and purifiedfrom CHO cells. Fusion proteins containing human Domain 1 (SEQ IDNO:115), Domains 1 and 2 (SEQ ID NO:116); or Domains 1, 2, and 3 (SEQ IDNO:117) were used in ELISA assays for determination of antibody binding.In some experiments, specificity of antibodies for human Siglec-8 wasevaluated in comparison with a fusion protein containing theextracellular Domains 1, 2, and 3 of Siglec-8 (SEQ ID NO:118) from theolive baboon (Papio anubis) (National Center for BiotechnologyInformation reference sequence XP_009193370.1).

For the antibody binding assays, ELISA plates (MaxiSorp; Nunc) werecoated overnight at 4° C. with fusion protein at 0.2 μg/ml and blockedfor 1 hour at room temperature with 2% BSA in PBS. Antibodies at 1 μg/mlwere added and the plates were incubated for 2 hours at roomtemperature. After washing the plates, horseradish peroxidase conjugatedsecondary antibody was added and the plates were incubated for 1 hour.Secondary antibodies were anti-human H+L HRP (Jackson ImmunoResearch,Catalog Number 709-035-149) for humanized antibodies or anti-mouse H+LHRP (Jackson ImmunoResearch, Catalog Number 715-035-151) for mouseantibodies. The plates were developed with TMB Substrate (Sigma, CatalogNumber T0440-1L).

The murine 2E2 antibody and the HEKA IgG1 antibody bound to the Domain 1fusion protein, indicating that the epitope for these two antibodiesresides in the N-terminal ligand-binding domain (Table 14). In contrast,murine 1C3 antibody bound to the Domain 1 and Domain 2 fusion proteinbut did not demonstrate detectable binding to the Domain 1 fusionprotein, indicating that the epitope for this antibody is in Domain 2(Table 14).

TABLE 14 Binding of anti-Siglec-8 antibodies to epitopes in humanSiglec-8 Domain 1-Fc Domain 1 + Domain 1 + (SEQ ID 2-Fc (SEQ 2 + 3-Fc(SEQ Epitope Antibody NO: 115) ID NO: 116) ID NO: 117) domain 2E2 + + +1 HEKA + + + 1 1C3 − + + 2

The murine 4F11 and 1H10 antibodies bound to human Siglec-8 and thepredicted Siglec-8 protein sequence from the olive baboon (Papio anubis)(National Center for Biotechnology Information reference sequenceXP_009193370.1). In ELISA screens of domain fusion proteins and onWestern blots of reduced SDS-PAGE gels, 4F11 recognized a linear epitopein human Siglec-8 Domain 1 and 1H10 recognized a linear epitope thatincluded sequences in human Siglec-8 Domain 3. 1C3 did not recognizedenatured sequences in human Siglec-8 Domain 2 indicating it recognizeda conformational epitope. Antibodies 4F11 and 1H10 show potent depletionof eosinophils from human peripheral blood leukocytes in the presence of50 ng/ml IL-5, with an EC₅₀ of 5.9 and 41 ng/ml, respectively (Table15). Murine 1C3 antibody and murine 2E2 antibody specific for humanSiglec-8 did not cross-react with baboon Siglec-8.

TABLE 15 Binding of anti-Siglec-8 antibodies to epitopes in human orbaboon Siglec-8 and depleting activity of the antibodies for humaneosinophils Epitope in Baboon Mean EC₅₀ human Linear epitope cross-(ng/ml) for Siglec-8 (reduced SDS- reactivity eosinophil killingAntibody domain PAGE) (ELISA) (2 donors) 4F11 1 + + 5.9 1H10 3 + + 41  1C3 2 − − 7.7 2E2 1 + − 6.9 Mean EC50 indicates mean half-maximalantibody concentrations required for eosinophil depletion from 2independent assays.

Binding of antibodies to human and baboon eosinophils was determined byflow cytometry. Human or baboon peripheral blood leukocyte preparationswere labeled with saturating amounts of anti-Siglec-8 monoclonalantibodies 2E2, 1C3, and 1H10 or mouse IgG1 isotype control antibody.Anti-Siglec-8 antibodies were visualized by a secondary anti-mouse IgGH+L AlexaFluor 647. Eosinophils were identified using primatecross-reactive monoclonal antibodies to CD49d and CD16 along with highgranularity scatter. Murine 1H10 antibody bound to baboon and humaneosinophils while mouse 2E2 and 1C3 antibodies bound to humaneosinophils but did not cross-react with baboon eosinophils (FIG. 10).These results were unexpected since other monoclonal mouse anti-Siglec-8antibodies that bind to human Siglec-8 have been shown to not recognizenon-human primate Siglec-8. See Hudson et al., J. Clin. Immunol., 2011,31(6):1045-53.

SEQUENCES Amino acid sequence of mouse 2E2 heavy chain variable domain(SEQ ID NO: 1)QVQLKESGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSVTVSSAmino acid sequence of 2E2 RHA heavy chain variable domain(SEQ ID NO: 2)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSAmino acid sequence of 2E2 RHB heavy chain variable domain(SEQ ID NO: 3)EVQLVESGGGLVQPGGSLRLSCAVSGFSLTIYGAHWVRQAPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSAmino acid sequence of 2E2 RHC heavy chain variable domain(SEQ ID NO: 4)EVQLVESGGGLVQPGGSLRLSCAVSGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSAmino acid sequence of 2E2 RHD heavy chain variable domain(SEQ ID NO: 5)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWLSVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSAmino acid sequence of 2E2 RHE heavy chain variable domain(SEQ ID NO: 6)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSAmino acid sequence of 2E2 RHF heavy chain variable domain(SEQ ID NO: 7)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSAmino acid sequence of 2E2 RHG heavy chain variable domain(SEQ ID NO: 8)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVSVIWAGGSTNYNSALMSRFSISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSAmino acid sequence of 2E2 RHA2 heavy chain variable domain(SEQ ID NO: 9)QVQLQESGPGLVKPSETLSLTCTVSGGSISIYGAHWIRQPPGKGLEWIGVIWAGGSTNYNSALMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGSSPYYYSMEYWGQGTLVTVSSAmino acid sequence of 2E2 RHB2 heavy chain variable domain(SEQ ID NO: 10)QVQLQESGPGLVKPSETLSLTCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKNQVSLKLSSVTAADTAVYYCARDGSSPYYYSMEYWGQGTLVTVSSAmino acid sequence of 2E2 RHE S-G mutant heavy chain variable domain(SEQ ID NO: 11)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYGMEYWGQGTTVTVSSAmino acid sequence of 2E2 RHE E-D heavy chain variable domain(SEQ ID NO: 12)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMDYWGQGTTVTVSSAmino acid sequence of 2E2 RHE Y-V heavy chain variable domain(SEQ ID NO: 13)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEVWGQGTTVTVSSAmino acid sequence of 2E2 RHE triple mutant heavy chain variable domain(SEQ ID NO: 14)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYGMDVWGQGTTVTVSSAmino acid sequence of mouse 2E2 light chain variable domain(SEQ ID NO: 15)QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKAmino acid sequence of 2E2 RKA light chain variable domain(SEQ ID NO: 16)EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKAmino acid sequence of 2E2 RKB light chain variable domain(SEQ ID NO: 17)EIILTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLWIYSTSNLASGVPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKAmino acid sequence of 2E2 RKC light chain variable domain(SEQ ID NO: 18)EIILTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKAmino acid sequence of 2E2 RKD light chain variable domain(SEQ ID NO: 19)EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLWIYSTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKAmino acid sequence of 2E2 RKE light chain variable domain(SEQ ID NO: 20)ETVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKAmino acid sequence of 2E2 RKF light chain variable domain(SEQ ID NO: 21)EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKAmino acid sequence of 2E2 RKG light chain variable domain(SEQ ID NO: 22)EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKAmino acid sequence of 2E2 RKA F-Y mutant light chain variable domain(SEQ ID NO: 23)EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPYTFGPGTKLDIKAmino acid sequence of 2E2 RKF F-Y mutant light chain variable domain(SEQ ID NO: 24)EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPYTFGPGTKLDIKAmino acid sequence of HEKA IgG1 heavy chain and HEKF IgG1 heavy chain(SEQ ID NO: 75)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGAmino acid sequence of HEKA kappa light chain (SEQ ID NO: 76)ETVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Amino acid sequence of HEKF kappa light chain(SEQ ID NO: 77)EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQRSSYPFTFGPGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAmino acid sequence of IgG1 heavy chain constant region (SEQ ID NO: 78)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Amino acid sequence of IgG4 heavy chain constant region(IgG4 contains a S228P mutation) (SEQ ID NO: 79)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG Amino acid sequence of Ig kappa light chain constantregion (SEQ ID NO: 80)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAmino acid sequence of murine 2C4 and 2E2 IgG1 heavy chain(SEQ ID NO: 81)QVQLKRASGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSH SPGAmino acid sequence of murine 2C4 kappa light chain (SEQ ID NO: 82)EIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECAmino acid sequence of murine 2E2 kappa light chain (SEQ ID NO: 83)QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Amino acid sequence of chimeric 2C4 and 2E2 IgG1heavy chain (SEQ ID NO: 84)QVQLKRASGPGLVAPSQSLSITCTVSGFSLTIYGAHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSISKDNSKSQVFLKINSLQTDDTALYYCARDGSSPYYYSMEYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGAmino acid sequence of chimeric 2C4 kappa light chain (SEQ ID NO: 85)EIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECAmino acid sequence of chimeric 2E2 kappa light chain (SEQ ID NO: 86)QIILTQSPAIMSASPGEKVSITCSATSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Amino acid sequence of HEKA IgG4 heavy chain(IgG4 contains a S228P mutation) (SEQ ID NO: 87)EVQLVESGGGLVQPGGSLRLSCAASGFSLTIYGAHWVRQAPGKGLEWVGVIWAGGSTNYNSALMSRFTISKDNSKNTVYLQMNSLRAEDTAVYYCARDGSSPYYYSMEYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGAmino acid sequence of mouse 1C3 heavy chain variabledomain (underlined residues comprise CDRs H1 and H2according to Chothia numbering) (SEQ ID NO: 106)EVQVVESGGDLVKSGGSLKLSCAASGFPFSSYAMSWVRQTPDKRLEWVAIISSGGSYTYYSDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARHETAQAAWFAYWGQGTLVTVSAAmino acid sequence of mouse 1H10 heavy chain variabledomain (underlined residues comprise CDRs H1 and H2according to Chothia numbering) (SEQ ID NO: 107)EVQLQQSGAELVRPGASVKLSCTASGFNIKDYYMYWVKQRPEQGLEWIGRIAPEDGDTEYAPKFQGKATVTADTSSNTAYLHLSSLTSEDTAVYYCTTEGNYYGSSILDYWGQGTTLTVSSAmino acid sequence of mouse 4F11 heavy chain variabledomain (underlined residues comprise CDRs H1 and H2according to Chothia numbering) (SEQ ID NO: 108)QVQLQQSGAELVKPGASVKISCKASGYAFRSSWMNWVKQRPGKGLEWIGQIYPGDDYTNYNGKFKGKVTLTADRSSSTAYMQLSSLTSEDSAVYFCARLGPYGPFADWGQGTLVTVSAAmino acid sequence of mouse 1C3 light chain variable domain(SEQ ID NO: 109)QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLAYGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFGGGTKLEIKAmino acid sequence of mouse 1H10 light chain variable domain(SEQ ID NO: 110)DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIYFTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKAmino acid sequence of mouse 4F11 light chain variable domain(SEQ ID NO: 111)QIVLTQSPAIVSASPGEKVTMTCSASSSVSYMYWYQQRPGSSPRLLIYDTSSLASGVPVRFSGSGSGTSYSLTISRIESEDAANYYCQQWNSDPYTFGGGTKLEIKAmino acid sequence of human Siglec-8 Domain 1 (SEQ ID NO: 112)MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRP Amino acid sequence of human Siglec-8 Domain 2(SEQ ID NO: 113)DILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVS Amino acid sequence of human Siglec-8 Domain 3(SEQ ID NO: 114)YPPWNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGAmino acid sequence of human Siglec-8 Domain 1 Fusion Protein(SEQ ID NO: 115)MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKAmino acid sequence of human Siglec-8 Domains 1 and 2 Fusion Protein(SEQ ID NO: 116)MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of human Siglec-8 Domains 1,2, and 3 Fusion Protein (SEQ ID NO: 117)MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAGDRPYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of baboon Siglec-8 Domains 1,2, and 3 Fusion Protein (SEQ ID NO: 118)MEGDRKYGDGYLLQVQELVTVQEGLCVHVPCSFSYPKDDWTYSDPVHGYWFRAGDRPYQEAPVATNNPDTEVQAETQGRFQLLGDRWSNDCSLSINDARKGDEGSYFFRLERGRMKWSYKSQLNYKAKQLSVFVTALTQRPDILIQGTLESGHPRNLTCSVPWACEQRMPPMISWIGTSVSSLGPITARFSVLTLIPKPQDHGTSLTCQVTLPGTGVTTTRTVQLDVSYPPWNLTVTVFQGDDTASTALGNGSSLSVLEGQSLRLVCAVDSNPPARLSWTRGSLTLCPSQPWNPGLLELLRVHVKDEGEFTCQAENPRGSQHISLSLSLQNEGTGTARPVSEVTLAAVGGIEGRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of baboon Siglec-8 Domains 1,2, and 3 (SEQ ID NO: 119)MEGDRKYGDGYLLQVQELVTVQEGLCVHVPCSFSYPKDDWTYSDPVHGYWFRAGDRPYQEAPVATNNPDTEVQAETQGRFQLLGDRWSNDCSLSINDARKGDEGSYFFRLERGRMKWSYKSQLNYKAKQLSVFVTALTQRPDILIQGTLESGHPRNLTCSVPWACEQRMPPMISWIGTSVSSLGPITARFSVLTLIPKPQDHGTSLTCQVTLPGTGVTTTRTVQLDVSYPPWNLTVTVFQGDDTASTALGNGSSLSVLEGQSLRLVCAVDSNPPARLSWTRGSLTLCPSQPWNPGLLELLRVHVKDEGEFTCQAENPRGSQHISLSLSLQNEGTGTARPVSEVTLAAVGG

1-76. (canceled)
 77. A method of treating or preventing a diseasemediated by cells expressing Siglec-8 in a subject, the methodcomprising administering to the subject an effective amount of ahumanized antibody that binds to a human Siglec-8, wherein the antibodycomprises a human IgG1 Fc region, and wherein at least one or two of theheavy chains of the antibody is/are non-fucosylated.
 78. The method ofclaim 77, wherein the disease is an eosinophil mediated-disease or amast cell mediated-disease.
 79. (canceled)
 80. The method of claim 77,wherein the antibody inhibits one or more symptoms of an allergicreaction.
 81. The method of claim 80, wherein the allergic reaction is aType I hypersensitivity reaction.
 82. The method of claim 77, whereinthe disease is selected from the group consisting of: asthma, allergicrhinitis, nasal polyposis, atopic dermatitis, chronic urticaria,mastocytosis, eosinophilic leukemia, and hypereosinophilic syndrome. 83.The method of claim 77, wherein the disease is selected from the groupconsisting of: pauci granulocytic asthma, acute or chronic airwayhypersensitivity, eosinophilic esophagitis, Churg-Strauss syndrome,inflammation associated with a cytokine, inflammation associated withcells expressing Siglec-8, malignancy associated with cells expressingSiglec-8, physical urticaria, cold urticaria, pressure-urticaria,bullous pemphigoid, food allergy, and allergic bronchopulmonaryaspergillosis (ABPA).
 84. The method of claim 77, wherein the subject issuffering from asthma that is not adequately controlled by an inhaledcorticosteroid, a short acting β2 agonist, a long acting β2 agonist, ora combination thereof. 85-91. (canceled)
 92. The method of claim 77,wherein the antibody is produced in a cell line having aalpha1,6-fucosyltransferase (Fut8) knockout.
 93. The method of claim 77,wherein the antibody is produced in a cell line overexpressingβ1,4-N-acetylglycosminyltransferase III (GnT-III).
 94. The method ofclaim 93, wherein the cell line additionally overexpresses Golgiμ-mannosidase II (ManII). 95-109. (canceled)
 110. The method of claim77, wherein the antibody comprises a heavy chain variable region and alight chain variable region, wherein the heavy chain variable regioncomprises (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:61,(ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:62, and(iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO:63; andwherein the light chain variable region comprises (i) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:64, (ii) HVR-L2 comprising theamino acid sequence of SEQ ID NO:65, and (iii) HVR-L3 comprising theamino acid sequence of SEQ ID NO:66.
 111. The method of claim 77,wherein the antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO:6; and a light chain variableregion comprising the amino acid sequence of SEQ ID NO:16 or
 21. 112.The method of claim 77, wherein the antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO:75 and a light chaincomprising the amino acid sequence of SEQ ID NO:76 or 77, or a heavychain comprising the amino acid sequence of SEQ ID NO:87 and a lightchain comprising the amino acid sequence of SEQ ID NO:76.
 113. Themethod of claim 77, wherein the antibody comprises a heavy chainvariable region comprising the amino acid sequence selected from SEQ IDNOs:2-14; and a light chain variable region comprising an amino acidsequence selected from the group consisting of SEQ ID NOs:16-24. 114.The method of claim 77, wherein the binding affinity or avidity of theantibody to a human Siglec-8 is higher than the binding affinity oravidity of antibody 2E2 or 2C4 to the human Siglec-8.
 115. The method ofclaim 77, wherein the antibody is administered to the subject in acomposition comprising the antibody and a pharmaceutically acceptablecarrier.
 116. The method of claim 115, wherein the antibody comprisesN-glycoside-linked carbohydrate chains linked to the Fc region, andwherein substantially none of the N-glycoside-linked carbohydrate chainscontain a fucose residue.
 117. The method of claim 92, wherein the cellline is a mammalian cell line.
 118. The method of claim 93, wherein thecell line is a mammalian cell line.
 119. The method of claim 77, whereinthe subject is a human.